scholarly journals BMP15 regulates the inhibin/activin system independently of ovulation rate control in sheep

Reproduction ◽  
2017 ◽  
Vol 153 (4) ◽  
pp. 395-404 ◽  
Author(s):  
Anthony Estienne ◽  
Belén Lahoz ◽  
Peggy Jarrier ◽  
Loys Bodin ◽  
José Folch ◽  
...  
Keyword(s):  
Granulosa Cells ◽  
Rate Control ◽  
Activin A ◽  
Ovulation Rate ◽  
Loss Of Function ◽  
Wild Type ◽  
Homozygous State ◽  
Gene Encoding ◽  
Morphogenetic Protein ◽  
Ovarian Inhibin

Polymorphisms in the gene encoding bone morphogenetic protein 15 (BMP15) have been associated with multiple ovulations in sheep. As BMP15 regulates inhibin expression in rodents, we assumed that the ovarian inhibin/activin system could mediate part of the effect of BMP15 mutations in the regulation of ovulation rate in sheep. To answer this question, we have studied the effects of two natural loss-of-function mutations of BMP15 on the expression of components of this system. The FecXR and the FecXGr mutations, when present respectively in Rasa Aragonesa ewes at the heterozygous state and in Grivette ewes at the homozygous state, were associated with a twofold increase in ovulation rate. There were only small differences between mutant and wild-type ewes for mRNA expression of INHA, INHBA, ACVR1B, ACVR2A, FST or TGFBR3 in granulosa cells and inhibin A or activin A concentrations in follicular fluid. Moreover, the effects of mutations differed between breeds. In cultures of granulosa cells from wild-type ewes, BMP15, acting alone or in synergy with GDF9, stimulated INHA, INHBA and FST expression, but inhibited the expression of TGFBR3. Activin A did not affect INHBA expression, but inhibited the expression of ACVR2A also. The complexity of the inhibin/activin system, including positive and antagonistic elements, and the differential regulation of these elements by BMP15 and activin can explain that the effects of BMP15 mutations differ when present in different genetic backgrounds. In conclusion, the ovarian inhibin/activin system is unlikely to participate in the increase of ovulation rate associated with BMP15 mutations in sheep.

scholarly journals Gonadotrophin-responsiveness of granulosa cells from bone morphogenetic protein 15 heterozygous mutant sheep

Reproduction ◽  
2009 ◽  
Vol 138 (3) ◽  
pp. 545-551 ◽  
Author(s):  
Kenneth P McNatty ◽  
Derek A Heath ◽  
Norma L Hudson ◽  
Stan Lun ◽  
Jennifer L Juengel ◽  
...  
Keyword(s):  
Bone Morphogenetic Protein ◽  
Granulosa Cells ◽  
Antral Follicle ◽  
Human Chorionic Gonadotrophin ◽  
Ovulation Rate ◽  
Wild Type ◽  
Binding Characteristics ◽  
Enhanced Sensitivity ◽  
Morphogenetic Protein ◽  
Bone Morphogenetic Protein 15

The aim of this study was to test the hypothesis that the higher ovulation-rate in ewes heterozygous for a mutation in bone morphogenetic protein 15 (BMP15; FecXI; otherwise known as Inverdale or I+ ewes) is due to granulosa cells developing an earlier responsiveness to LH, but not FSH. To address this hypothesis, granulosa cells were recovered from every individual nonatretic antral follicle (>2.5 mm diameter) from I+ and wild-type (++) ewes during anoestrus and the luteal and follicular phases and tested for their responsiveness to FSH and human chorionic gonadotrophin (hCG; a surrogate for LH). For the FSH receptor (FSHR) binding study, granulosa cells were harvested in three separate batches from all antral follicles (≥2.5 mm diameter) from I+ and ++ ewes. Using a highly-purified ovine FSH preparation, no evidence was found to suggest that I+ ewes have a higher ovulation-rate due to enhanced sensitivity of granulosa cells to FSH with respect to cAMP responsiveness or to their FSHR binding characteristics (equilibrium Kd or Bmax). By contrast, a significantly higher proportion of follicles from I+ ewes contained granulosa cells responsive to hCG. The higher proportion was due to cells from more small follicles (i.e. >2.5–4.5 mm diameter) developing a response to hCG. It is concluded that the mutation in the BMP15 gene in I+ ewes leads to an earlier acquisition of LH responsiveness by granulosa cells in a greater proportion of follicles and this accounts for the small but significantly higher ovulation-rate in these animals.

scholarly journals Activins regulate 17β-hydroxysteroid dehydrogenase type I transcription in murine gonadotrope cells

2009 ◽  
Vol 201 (1) ◽  
pp. 89-104 ◽  
Author(s):  
Beata Bak ◽  
Laura Carpio ◽  
Jinjing L Kipp ◽  
Pankaj Lamba ◽  
Ying Wang ◽  
...  
Keyword(s):  
Granulosa Cells ◽  
Intracellular Signaling ◽  
Cell Types ◽  
Hydroxysteroid Dehydrogenase ◽  
Activin A ◽  
Type I ◽  
Promoter Regions ◽  
Gene Encoding ◽  
17Β Estradiol ◽  
Smad Binding Element

Activins are pleiotropic members of the TGFβ superfamily and were initially characterized based on their abilities to stimulate FSH synthesis and secretion by gonadotrope cells of the anterior pituitary gland. Here, we identified the gene encoding the steroidogenic enzyme, 17β-hydroxysteroid dehydrogenase type I (17β-HSD1; Hsd17b1), as an activin-responsive gene in immortalized gonadotrope cells, LβT2. 17β-HSD1 catalyzes the conversion of estrone to the more active 17β-estradiol, and activin A stimulated an increase in this enzymatic activity in these cells. We demonstrated that activins signaled via the type I receptor, activin receptor-like kinase (ALK4), and the intracellular signaling protein, SMAD2, to regulate Hsd17b1 transcription in immediate-early fashion. Critical cis-elements, including a minimal SMAD-binding element, were mapped to within 100 bp of the start of transcription. Activin/ALK4 signaling also regulated Hsd17b1 transcription in both immortalized and primary cultured murine granulosa cells. The promoter regions mediating basal and activin/ALK4-regulated promoter activity were generally conserved across the different cell types. The data show that activin A rapidly regulates Hsd17b1 transcription in gonadotrope and granulosa cells and may thereby regulate local 17β-estradiol synthesis.

scholarly journals Effects of immunizing ewes against bone morphogenetic protein 15 on their responses to exogenous gonadotrophins to induce multiple ovulations

Reproduction ◽  
2011 ◽  
Vol 142 (4) ◽  
pp. 565-572 ◽  
Author(s):  
Jennifer L Juengel ◽  
Laurel D Quirke ◽  
Stan Lun ◽  
Derek A Heath ◽  
Peter D Johnstone ◽  
...  
Keyword(s):  
Bone Morphogenetic Protein ◽  
Granulosa Cells ◽  
Ovulation Rate ◽  
Keyhole Limpet Haemocyanin ◽  
Treatment Groups ◽  
Morphogenetic Protein ◽  
Water Based ◽  
Bone Morphogenetic Protein 15 ◽  
And Control ◽  
Inactivating Mutation

Sheep with a heterozygous inactivating mutation in the bone morphogenetic protein 15 (BMP15) gene experience an increased ovulation rate during either a natural oestrous cycle or a cycle in which exogenous FSH and eCG (gonadotrophins) are given to induce multiple ovulations. The primary aim of these studies was to determine whether ewes immunised against BMP15 would also show an improved superovulation rate following exogenous gonadotrophin treatment. A secondary aim was to determine the effects of BMP15 immunisation on ovarian follicular characteristics. In most ewes (i.e. >75%) immunised with a BMP15-keyhole limpet haemocyanin peptide in an oil-based adjuvant in order to completely neutralise BMP15 bioactivity, there was no superovulation response to exogenous gonadotrophins. In ewes treated with exogenous gonadotrophins following a BMP15-BSA peptide immunisation in a water-based adjuvant to partially neutralise BMP15 bioactivity, the ovulation rate response was similar to the control superovulation treatment groups. Characterisation of follicular function revealed that the water-based BMP15-immunised animals had fewer non-atretic follicles 2.5–3.5 or >4.5 mm in diameter compared with controls. Basal concentrations of cAMP were higher in granulosa cells from animals immunised against BMP15 than control animals. There were no significant differences in the concentrations of cAMP between granulosa cells from BMP15- and control-immunised animals when given FSH or hCG, although there were differences in the proportions of follicles in different size classes that responded to FSH or hCG. Thus, immunisation against BMP15 may have been causing premature luteinisation and thereby limiting the numbers of follicles recruited for ovulation following treatment with exogenous gonadotrophins.

scholarly journals Homozygote loss-of-function variants in the human COCH gene underlie hearing loss

2020 ◽  
Author(s):  
Nada Danial-Farran ◽  
Elena Chervinsky ◽  
Prathamesh Thangaraj Nadar-Ponniah ◽  
Eran Cohen Barak ◽  
Shahar Taiber ◽  
...  
Keyword(s):  
Hearing Loss ◽  
Autosomal Dominant ◽  
Dominant Negative ◽  
Dominant Negative Effect ◽  
Loss Of Function ◽  
Wild Type ◽  
Cos7 Cell ◽  
Gene Encoding ◽  
Negative Effect ◽  
Syndromic Hearing Loss

AbstractSince 1999, the COCH gene encoding cochlin, has been linked to the autosomal dominant non-syndromic hearing loss, DFNA9, with or without vestibular abnormalities. The hearing impairment associated with the variants affecting gene function has been attributed to a dominant-negative effect. Mutant cochlin was seen to accumulate intracellularly, with the formation of aggregates both inside and outside the cells, in contrast to the wild-type cochlin that is normally secreted. While an additional recessive variant in the COCH gene (DFNB110) has recently been reported, the mechanism of the loss-of-function (LOF) effect of the COCH gene product remains unknown. In this study, we used COS7 cell lines to investigate the consequences of a novel homozygous frameshift variant on RNA transcription, and on cochlin translation. Our results indicate a LOF effect of the variant and a major decrease in cochlin translation. This data has a dramatic impact on the accuracy of genetic counseling for both heterozygote and homozygote carriers of LOF variants in COCH.

Selective binding of RGMc/hemojuvelin, a key protein in systemic iron metabolism, to BMP-2 and neogenin

2008 ◽  
Vol 294 (4) ◽  
pp. C994-C1003 ◽  
Author(s):  
Robin Kuns-Hashimoto ◽  
David Kuninger ◽  
Mahta Nili ◽  
Peter Rotwein
Keyword(s):  
Signaling Proteins ◽  
Wild Type ◽  
Single Chain ◽  
Gene Encoding ◽  
Morphogenetic Protein ◽  
Biochemical Assays ◽  
Juvenile Hemochromatosis ◽  
Repulsive Guidance Molecule ◽  
Membrane Spanning ◽  
Guidance Molecule

Juvenile hemochromatosis is a severe and rapidly progressing hereditary disorder of iron overload, and it is caused primarily by defects in the gene encoding repulsive guidance molecule c/hemojuvelin (RGMc/HJV), a recently identified protein that undergoes a complicated biosynthetic pathway in muscle and liver, leading to cell membrane-linked single-chain and heterodimeric species, and two secreted single-chain isoforms. RGMc modulates expression of the hepatic iron regulatory factor, hepcidin, potentially through effects on signaling by the bone morphogenetic protein (BMP) family of soluble growth factors. To date, little is known about specific pathogenic defects in disease-causing RGMc/HJV proteins. Here we identify functional abnormalities in three juvenile hemochromatosis-linked mutants. Using a combination of approaches, we first show that BMP-2 could interact in biochemical assays with single-chain RGMc species, and also could bind to cell-associated RGMc. Two mouse RGMc amino acid substitution mutants, D165E and G313V (corresponding to human D172E and G320V), also could bind BMP-2, but less effectively than wild-type RGMc, while G92V (human G99V) could not. In contrast, the membrane-spanning protein, neogenin, a receptor for the related molecule, RGMa, preferentially bound membrane-associated heterodimeric RGMc and was able to interact on cells only with wild-type RGMc and G92V. Our results show that different isoforms of RGMc/HJV may play unique physiological roles through defined interactions with distinct signaling proteins and demonstrate that, in some disease-linked RGMc mutants, these interactions are defective.

Involvement of OpsLTP1 from Opuntia streptacantha in abiotic stress adaptation and lipid metabolism

2019 ◽  
Vol 46 (9) ◽  
pp. 816
Author(s):  
Mario Rojas ◽  
Francisco Jimenez-Bremont ◽  
Claudia Villicaña ◽  
Laura Carreón-Palau ◽  
Bertha Olivia Arredondo-Vega ◽  
...  
Keyword(s):  
Abiotic Stress ◽  
Stress Responses ◽  
Germination Rate ◽  
Lower Amount ◽  
Lipid Transfer ◽  
Loss Of Function ◽  
Wild Type ◽  
Biotic And Abiotic Stress ◽  
Gene Encoding

Plant lipid transfer proteins (LTPs) exhibit the ability to transfer lipids between membranes in vitro, and have been implicated in diverse physiological processes associated to plant growth, reproduction, development, biotic and abiotic stress responses. However, their mode of action is not yet fully understood. To explore the functions of the OpsLTP1 gene encoding a LTP from cactus pear Opuntia streptacantha Lem., we generated transgenic Arabidopsis thaliana (L.) Heynh. plants to overexpress OpsLTP1 and contrasted our results with the loss-of-function mutant ltp3 from A. thaliana under abiotic stress conditions. The ltp3 mutant seeds showed impaired germination under salt and osmotic treatments, in contrast to OpsLTP1 overexpressing lines that displayed significant increases in germination rate. Moreover, stress recovery assays showed that ltp3 mutant seedlings were more sensitive to salt and osmotic treatments than wild-type plants suggesting that AtLTP3 is required for stress-induced responses, while the OpsLTP1 overexpressing line showed no significant differences. In addition, OpsLTP1 overexpressing and ltp3 mutant seeds stored lower amount of total lipids compared with wild-type seeds, showing changes primarily on 16C and 18C fatty acids. However, ltp3 mutant also lead changes in lipid profile and no over concrete lipids which may suggest a compensatory activation of other LTPs. Interestingly, linoleic acid (18:2ω6) was consistently increased in neutral, galactoglycerolipids and phosphoglycerolipids of OpsLTP1 overexpressing line indicating a role of OpsLTP1 in the modulation of lipid composition in A. thaliana.

Effects of an Autoregulatory Senescence-inhibitor Gene Construct on Nicotiana alata Link and Otto.

HortScience ◽  
1998 ◽  
Vol 33 (3) ◽  
pp. 519d-519 ◽  
Author(s):  
Kenneth R. Schroeder ◽  
Dennis P. Stimart
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Plant Height ◽  
Dry Weight ◽  
Nicotiana Alata ◽  
Wild Type ◽  
Gene Encoding ◽  
Delayed Senescence ◽  
Gene Construct ◽  
Shoot Dry Weight ◽  
Inhibitor System

Nicotiana alata Link and Otto. was transformed via Agrobacterium tumefaciens encoding a senescence-specific promoter SAG12 cloned from Arabidopsis thaliana fused to a Agrobacterium tumefaciens gene encoding isopentenyl transferase (IPT) that catalyzes cytokinin synthesis. This was considered an autoregulatory senescence-inhibitor system. In 1996, we reported delayed senescence of intact flowers by 2 to 6 d and delayed leaf senescence of transgenic vs. wild-type N. alata. Further evaluations in 1997 revealed several other interesting effects of the SAG12-IPT gene construct. Measurement of chlorophyll content of mature leaves showed higher levels of both chlorophyll a and b in transgenic material under normal fertilization and truncated fertilization regimes. At 4 to 5 months of age transgenic plants expressed differences in plant height, branching, and dry weight. Plant height was reduced by 3 to 13 cm; branch counts increased 2 to 3 fold; and shoot dry weight increased up to 11 g over wild-type N. alata. These observations indicate the system is not tightly autoregulated and may prove useful to the floriculture industry for producing compact and more floriferous plants.

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

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.

scholarly journals The NALCN Channel Regulator UNC-80 Functions in a Subset of Interneurons To Regulate Caenorhabditis elegans Reversal Behavior

2019 ◽  
Vol 10 (1) ◽  
pp. 199-210 ◽  
Author(s):  
Chuanman Zhou ◽  
Jintao Luo ◽  
Xiaohui He ◽  
Qian Zhou ◽  
Yunxia He ◽  
...  
Keyword(s):  
Plant Hormone ◽  
Neuronal Excitability ◽  
Resting Membrane Potential ◽  
Loss Of Function ◽  
Wild Type ◽  
C Elegans ◽  
Link Type ◽  
Complex Functions ◽  
And Function ◽  
Multiple Loss

NALCN (Na+leak channel, non-selective) is a conserved, voltage-insensitive cation channel that regulates resting membrane potential and neuronal excitability. UNC79 and UNC80 are key regulators of the channel function. However, the behavioral effects of the channel complex are not entirely clear and the neurons in which the channel functions remain to be identified. In a forward genetic screen for C. elegans mutants with defective avoidance response to the plant hormone methyl salicylate (MeSa), we isolated multiple loss-of-function mutations in unc-80 and unc-79. C. elegans NALCN mutants exhibited similarly defective MeSa avoidance. Interestingly, NALCN, unc-80 and unc-79 mutants all showed wild type-like responses to other attractive or repelling odorants, suggesting that NALCN does not broadly affect odor detection or related forward and reversal behaviors. To understand in which neurons the channel functions, we determined the identities of a subset of unc-80-expressing neurons. We found that unc-79 and unc-80 are expressed and function in overlapping neurons, which verified previous assumptions. Neuron-specific transgene rescue and knockdown experiments suggest that the command interneurons AVA and AVE and the anterior guidepost neuron AVG can play a sufficient role in mediating unc-80 regulation of the MeSa avoidance. Though primarily based on genetic analyses, our results further imply that MeSa might activate NALCN by direct or indirect actions. Altogether, we provide an initial look into the key neurons in which the NALCN channel complex functions and identify a novel function of the channel in regulating C. elegans reversal behavior through command interneurons.

scholarly journals Gain-of-function GABRB3 variants identified in vigabatrin-hypersensitive epileptic encephalopathies

2020 ◽  
Vol 2 (2) ◽  
Author(s):  
Nathan L Absalom ◽  
Vivian W Y Liao ◽  
Kavitha Kothur ◽  
Dinesh C Indurthi ◽  
Bruce Bennetts ◽  
...  
Keyword(s):  
De Novo ◽  
Drug Effects ◽  
Receptor Expression ◽  
Aminobutyric Acid ◽  
Loss Of Function ◽  
Molecular Phenotype ◽  
Gain Of Function ◽  
Gene Encoding ◽  
Epileptic Encephalopathies ◽  
Receptor Phenotype

Abstract Variants in the GABRB3 gene encoding the β3-subunit of the γ-aminobutyric acid type A ( receptor are associated with various developmental and epileptic encephalopathies. Typically, these variants cause a loss-of-function molecular phenotype whereby γ-aminobutyric acid has reduced inhibitory effectiveness leading to seizures. Drugs that potentiate inhibitory GABAergic activity, such as nitrazepam, phenobarbital or vigabatrin, are expected to compensate for this and thereby reduce seizure frequency. However, vigabatrin, a drug that inhibits γ-aminobutyric acid transaminase to increase tonic γ-aminobutyric acid currents, has mixed success in treating seizures in patients with GABRB3 variants: some patients experience seizure cessation, but there is hypersensitivity in some patients associated with hypotonia, sedation and respiratory suppression. A GABRB3 variant that responds well to vigabatrin involves a truncation variant (p.Arg194*) resulting in a clear loss-of-function. We hypothesized that patients with a hypersensitive response to vigabatrin may exhibit a different γ-aminobutyric acid A receptor phenotype. To test this hypothesis, we evaluated the phenotype of de novo variants in GABRB3 (p.Glu77Lys and p.Thr287Ile) associated with patients who are clinically hypersensitive to vigabatrin. We introduced the GABRB3 p.Glu77Lys and p.Thr287Ile variants into a concatenated synaptic and extrasynaptic γ-aminobutyric acid A receptor construct, to resemble the γ-aminobutyric acid A receptor expression by a patient heterozygous for the GABRB3 variant. The mRNA of these constructs was injected into Xenopus oocytes and activation properties of each receptor measured by two-electrode voltage clamp electrophysiology. Results showed an atypical gain-of-function molecular phenotype in the GABRB3 p.Glu77Lys and p.Thr287Ile variants characterized by increased potency of γ-aminobutyric acid A without change to the estimated maximum open channel probability, deactivation kinetics or absolute currents. Modelling of the activation properties of the receptors indicated that either variant caused increased chloride flux in response to low concentrations of γ-aminobutyric acid that mediate tonic currents. We therefore propose that the hypersensitivity reaction to vigabatrin is a result of GABRB3 variants that exacerbate GABAergic tonic currents and caution is required when prescribing vigabatrin. In contrast, drug strategies increasing tonic currents in loss-of-function variants are likely to be a safe and effective therapy. This study demonstrates that functional genomics can explain beneficial and adverse anti-epileptic drug effects, and propose that vigabatrin should be considered in patients with clear loss-of-function GABRB3 variants.

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