Distinct expression patterns of TLR transcripts in human oocytes and granulosa cells from primordial and primary follicles.

Ovulation has long been regarded as a process resembling an inflammatory response. Previously, luteinizing hormone (LH) was shown to induce Toll-like receptor 2 (TLR2) and TLR4 in granulosa cells from preovulatory hormone-dependent follicles. However, whether this could already initiate before the hormone-dependent phase is currently unknown. The aim of this study was to investigate TLR genes in human oocytes and granulosa cells from primordial and primary ovarian follicles during the hormone-independent phase. A class-comparison study of existing oocyte and granulosa cell RNA sequencing transcriptomes from primordial (n = 539 follicles) and primary (n = 261) follicles collected from three patients was examined. This revealed a distinct expression pattern of TLR3, TLR4 and TLR5 transcripts. Interestingly, the TLR3 protein was differentially detected in both the oocyte and the granulosa cells in primordial and primary follicles, suggesting that TLR3 is maternally contributed both as mRNA and protein. Intracellularly, the compartmentalized TLR3 dot-like staining in the intersection between the oocyte and the surrounding primordial granulosa cells. The TLR4 protein was detected in both primordial and primary follicles, with a notable staining in the granulosa cells. We functionally challenged ovaries in vitro, by polyinosinic:polycytidylic acid (poly I:C) and LPS, known to activate TLR3 and TLR4, respectively, and found a tendency for increased IL-6 production, which was particular evident in the LPS-treated group. Based on the expression of TLRs, it is notably that human primordial and primary follicles express genes that would allow them to respond to innate immune proteins and cytokines during follicle activation.

Transcripts encoding free radical scavengers in human granulosa cells from primordial and primary ovarian follicles.

PURPOSE: To study the presence and distribution of genes encoding free radical scavengers in human granulosa cells from primordial and primary ovarian follicles. METHODS: A class comparison study on existing granulosa cell transcriptome from primordial (n = 539 follicles) and primary (n = 261) follicles donated by three women having ovarian tissue cryopreserved before chemotherapy was performed and interrogated. RESULTS: In granulosa cells from primordial follicles, 30 genes were annotated 'mitochondrial dysfunction' including transcripts (PRDX5, TXN2) encoding enzymatic free radical scavengers peroxiredoxin 5 and thioredoxin 2. Several apoptosis regulation genes were noted (BCL2, CAS8, CAS9, AIFM1). In granulosa cells from primary follicles, mitochondrial dysfunction signalling pathway was annotated. High expression of transcripts encoding the free radical scavenger peroxiredoxin 3, as well as anti-apoptotic enzyme BCL2, was found. Interestingly, PARK7 encoding the deglycase (DJ-1) protein was expressed in granulosa cells from primary follicles. DJ-1 is implicated in oxidative defence and functions as a positive regulator of the androgen receptor and as a negative regulator of the phosphatidylinositol 3-kinase (PI3K)/phosphatase and tensin homolog (PTEN)/serine-threonine protein kinase (AKT) signalling pathway suppressor PTEN. CONCLUSIONS: The results indicate extensive energy production and free radical scavenging in the granulosa cells of primordial follicles with potential implications for ovarian ageing, cigarette smoking, premature ovarian failure and polycystic ovarian syndrome. Furthermore, DJ-1 may be involved in androgen responsiveness and the regulation of follicle growth via PI3K/PTEN/AKT signalling pathway regulation in the granulosa cells of primary follicles. The involvement of mitochondrial free radical production in the age-related decline of competent oocytes is becoming apparent.

Granulosa cells from human primordial and primary follicles show differential global gene expression profiles.

STUDY QUESTION: Can novel genetic candidates involved in follicle dormancy, activation and integrity be identified from transcriptomic profiles of isolated granulosa cells from human primordial and primary follicles? SUMMARY ANSWER: The granulosa cell compartment of the human primordial and primary follicle was extensively enriched in signal transducer and activator of transcription 3 (STAT3) and cAMP-response element binding protein (CREB) signalling, and several other putative signalling pathways that may also be mediators of follicle growth and development were identified. WHAT IS KNOWN ALREADY: Mechanistic target of rapamycin kinase (mTOR) signalling and the factors Forkhead Box L2 (FOXL2) and KIT proto-oncogene receptor tyrosine kinase (KITL) may be involved in defining the early steps of mammalian follicular recruitment through complex bidirectional signalling between the oocyte and granulosa cells. cAMP/protein kinase K (PKA)/CREB signalling is a feature of FSH-induced regulation of granulosa cell steroidogenesis that is essential to normal human fertility. STUDY DESIGN, SIZE, DURATION: A class comparison study was carried out on primordial follicles (n = 539 follicles) and primary follicles (n = 261) follicles) donated by three women having ovarian tissue cryopreserved before chemotherapy. PARTICIPANTS/MATERIALS, SETTING, METHODS: RNA samples from isolates of laser capture micro-dissected oocytes and follicles from the primordial and primary stage, respectively, were sequenced on the HiSeq Illumina platform. Data mapping, quality control, filtering, FPKM (fragments per kilobase of exon per million) normalization and comparisons were performed. The granulosa cell contribution in whole follicle isolates was extracted in silico. Modelling of complex biological systems was performed using Ingenuity Pathway Analysis (IPA). For validation of transcriptomic findings, we performed quantitative RT-PCR of selected candidate genes. Furthermore, we interrogated the in situ localization of selected corresponding proteins using immunofluorescence. MAIN RESULTS AND THE ROLE OF CHANCE: Our differentially expressed gene analysis revealed a number of transcripts in the granulosa cells to be significantly down- (736 genes) or up- (294 genes) regulated during the human primordial-to-primary follicle transition. The IPA analysis revealed enriched canonical signalling pathways not previously associated with granulosa cells from human primordial and primary follicles. Immunofluorescent staining of human ovarian tissue explored the intra-ovarian localization of FOG2, and FOXL2, which revealed the presence of forkhead box L2 (FOXL2) in both oocytes and granulosa cells in primary follicles, with a more enriched staining in the granulosa cells in primary follicles. Friend of GATA 2 (FOG2) stained strongly in oocytes in primordial follicles, with a shift towards granulosa cell as follicle stage advanced. LARGE SCALE DATA: http://users-birc.au.dk/biopv/published_data/ernst_et_al_GC_2017/. LIMITATIONS REASONS FOR CAUTION: This is a descriptive study, and no functional assays were employed. The study was based on a limited number of patients, and it is acknowledged that natural biological variance exists in human samples. Strict filters were applied to accommodate the in silico extraction of the granulosa cell contribution. In support of this, quantitative RT-PCR was used to confirm selected candidate genes, and immunofluorescent staining was employed to interrogate the intra-ovarian distribution of selected corresponding proteins. Moreover, it is unknown whether the primordial follicles analysed represent those still in the resting pool, or those from the cohort that have entered the growing pool. WIDER IMPLICATIONS OF THE FINDINGS: We present, for the first time, a detailed description of global gene activity in the human granulosa cell compartment of primordial and primary follicles. These results may be utilized in the development of novel clinical treatment strategies aimed at improving granulosa cell function. STUDY FUNDING/COMPETING INTEREST(S): E.H.E. was supported by the Health Faculty, Aarhus University and Kong Christian Den Tiendes Fond. K.L.H. was supported by a grant from Fondens til Lægevidenskabens Fremme and Kong Christian Den Tiendes Fond. No authors have competing interests to declare.

Dormancy and activation of human oocytes from primordial and primary follicles: molecular clues to oocyte regulation.

STUDY QUESTION: Do specific transcriptome dynamics in human oocytes from primordial and primary follicles identify novel pathways in oocyte activation? SUMMARY ANSWER: The transcriptomic profiles in oocytes from primordial and primary follicles, respectively, revealed several new canonical pathways as putative mediators of oocyte dormancy and activation. WHAT IS KNOWN ALREADY: Cellular signaling pathways including PI3K/AKT and AKT/mTOR as well as TGF-ß and IGF signaling are known to regulate the primordial-to-primary transition in mammalian follicle development. STUDY DESIGN, SIZE, DURATION: We performed a class comparison study on human oocytes from primordial (n = 436) and primary (n = 182) follicles donated by three women having ovarian tissue cryopreserved before chemotherapy. PARTICIPANTS/MATERIALS, SETTING, METHODS: RNA was extracted from oocytes from primordial and primary follicles isolated by Laser Capture Microdissection, and submitted to the HiSeq Illumina platform. Data mapping, quality control, filtering and expression analysis were performed using Tophat (2.0.4), Cufflinks (2.0.2), BWA (0.6.2) and software R. Modeling of complex biological systems was performed using the IPA® software. Finally, qPCR and immunohistochemistry were employed to explore expression and localization of selected genes and products in human ovarian tissue. MAIN RESULTS AND THE ROLE OF CHANCE: We found 223 and 268 genes down-regulated and up-regulated, respectively, in the oocytes during the human primordial-to-primary follicle transition (P < 0.05 and/or FPKM fold-change >2). IPA® enrichment analysis revealed known pathways ('mTOR Signaling', 'PI3K/AKT Signaling' and 'PTEN Signaling') as well as enriched canonical pathways not previously associated with human ovarian follicle development such as 'ErB Signaling' and 'NGF Signaling' in the down-regulated category and 'Regulation of eIF4 and P70S6K Signaling' and 'HER-2 Signaling in Breast Cancer' in the up-regulated group. Additionally, immunohistochemistry on human ovarian tissue explored the intraovarian localization of VASA, FOXO1 and eIF4E. LARGE SCALE DATA: http://users-birc.au.dk/biopv/published_data/ernst_2017/. LIMITATIONS, REASONS FOR CAUTION: This is a descriptive analysis and no functional studies were performed. The study was based on a limited number of patients and the experimental design could not take into account the natural biological variance in human samples. Therefore, qPCR was used to confirm selected genes alongside immunohistochemical stainings. WIDER IMPLICATIONS OF THE FINDINGS: This study shows, for the first time, a detailed molecular description of global gene transcription activities in oocytes from primordial and primary follicles, respectively. Knowing the global transcription profiles of human oocyte dormancy and activation are important in developing new clinical applications. STUDY FUNDING/COMPETING INTEREST(S): E.H.E. was supported by Health Faculty, Aarhus University and Kong Christian Den Tiendes Fond. K.H. and S.F. were supported by an MRC (UK) project grant MR/M012638/1. K.L.H. was supported by grants from Fonden til Lægevidenskabens Fremme, Kong Christian Den Tiendes Fond. K.L.H. and L.S. were supported by the IDEAS grant from Aarhus University Research Foundation (AUFF). There are no conflicts of interest.

Direct Observation of Enhanced Nitric Oxide in a Murine Model of Diabetic Nephropathy.

Uncoupling of nitric oxide synthase (NOS) secondary to redox signaling is a central mechanism in endothelial and macrophage activation. To date studies on the production of nitric oxide (NO) during the development of diabetic complications show paradoxical results. We previously showed that recoupling eNOS by increasing the eNOS cofactor tetrahydrobiopterin (BH4) could restore endothelial function and prevent kidney injury in experimental kidney transplantation. Here, we employed a diabetic mouse model to investigate the effects of diabetes on renal tissue NO bioavailability. For this, we used in vivo NO trapping, followed by electron paramagnetic resonance spectroscopy. In addition, we investigated whether coupling of NOS by supplying the cofactor BH4 could restore glomerular endothelial barrier function. Our data show that overall NO availability at the tissue level is not reduced sixteen weeks after the induction of diabetes in apoE knockout mice, despite the presence of factors that cause endothelial dysfunction, and the presence of the endogenous NOS inhibitor ADMA. Targeting uncoupled NOS with the BH4 precursor sepiapterin further increases NO availability, but did not modify renal glomerular injury. Notably, glomerular heparanase activity as a driver for loss of glomerular barrier function was not reduced, pointing towards NOS-independent mechanisms. This was confirmed by unaltered increased glomerular presence of cathepsin L, the protease that activates heparanase.

Identification of free nitric oxide radicals in rat bone marrow: implications for progenitor cell mobilization in hypertension.

Nitric oxide (NO) has been implicated in matrix metallopeptidase 9 (MMP9)-dependent mobilization of hematopoietic stem and progenitor cells from bone marrow (BM). However, direct measurement of NO in the BM remained elusive due to its low in situ concentration and short lifetime. Using NO spin trapping and electron paramagnetic resonance (EPR) spectroscopy we give the first experimental confirmation of free NO radicals in rodent BM. NO production was quantified and attributed to enzymatic activity of NO synthases (NOS). Although endothelial NOS (eNOS) accounts for most (66%) of basal NO, we identified a significant contribution (23%) from inducible NOS (iNOS). Basal NO levels closely correlate with MMP9 bioavailability in BM of both hypertensive and control rats. Our observations support the hypothesis that inadequate mobilization of BM-derived stem and progenitor cells in hypertension results from impaired NOS/NO/MMP9 signalling in BM, a condition that may be corrected with pharmacological intervention.

Global gene analysis of oocytes from early stages in human folliculogenesis shows high expression of novel genes in reproduction.

The pool of primordial follicles in humans is laid down during embryonic development and follicles can remain dormant for prolonged intervals, often decades, until individual follicles resume growth. The mechanisms that induce growth and maturation of primordial follicles are poorly understood but follicles once activated either continue growth or undergo atresia. We have isolated pure populations of oocytes from human primordial, intermediate and primary follicles using laser capture micro-dissection microscopy and evaluated the global gene expression profiles by whole-genome microarray analysis. The array data were confirmed by qPCR for selected genes. A total of 6301 unique genes were identified as significantly expressed representing enriched specific functional categories such as 'RNA binding', 'translation initiation' and 'structural molecule activity'. Several genes, some not previously known to be associated with early oocyte development, were identified with exceptionally high expression levels, such as the anti-proliferative transmembrane protein with an epidermal growth factor-like and two follistatin-like domains (TMEFF2), the Rho-GTPase-activating protein oligophrenin 1 (OPHN1) and the mitochondrial-encoded ATPase6 (ATP6). Thus, the present study provides not only a technique to capture and perform transcriptome analysis of the sparse material of human oocytes from the earliest follicle stages but further includes a comprehensive basis for our understanding of the regulatory factors and pathways present during early human folliculogenesis.

NO formation by neuronal NO-synthase can be controlled by ultrafast electron injection from a nanotrigger.

Nitric oxide synthases (NOSs) are unique flavohemoproteins with various roles in mammalian physiology. Constitutive NOS catalysis is initiated by fast hydride transfer from NADPH, followed by slower structural rearrangements. We used a photoactive nanotrigger (NT) to study the initial electron transfer to FAD in native neuronal NOS (nNOS) catalysis. Molecular modeling and fluorescence spectroscopy showed that selective NT binding to NADPH sites close to FAD is able to override Phe1395 regulation. Ultrafast injection of electrons into the protein electron pathway by NT photoactivation through the use of a femtosecond laser pulse is thus possible. We show that calmodulin, required for NO synthesis by constitutive NOS, strongly promotes intramolecular electron flow (6.2-fold stimulation) by a mechanism involving proton transfer to the reduced FAD(-) site. Site-directed mutagenesis using the S1176A and S1176T mutants of nNOS supports this hypothesis. The NT synchronized the initiation of flavoenzyme catalysis, leading to the formation of NO, as detected by EPR. This NT is thus promising for time-resolved X-ray and other cellular applications.

Magnetic resonance imaging of phase transitions in nitinol.

Magnetic resonance images are prone to artifacts caused by metallic objects. Apart from being a source of image degradation, such artifacts can also provide information about the magnetic properties of the foreign object. In this work, we aim to explore the potential of magnetic resonance imaging to detect and characterize changes in magnetic properties of nitinol undergoing temperature- or strain-induced phase changes. A spin echo and a gradient echo method were used to measure the magnetization changes related to the phase transformations. Results of both methods were in agreement and in accordance with the independent measurements using a vibrating sample magnetometer. Magnetic resonance imaging turned out to be a suitable method to visualize and quantify magnetization and phase changes in situ. It is not restricted to a single imaging strategy and does not require any modification of the test object. The results indicate the potential of magnetic resonance imaging to provide direct feedback of the thermomechanical state of the alloy.

Magnetic resonance imaging of microstructure transition in stainless steel.

Magnetic resonance images are prone to artifacts caused by metallic objects. Such artifacts may not only hamper image interpretation, but also have been shown to provide information about the magnetic properties of the substances involved. In this work, we aim to explore the potential of MRI to detect, localize and characterize changes in magnetic properties that may occur when certain alloys have been exposed to a thermomechanical stress. For this purpose, stainless steel 304 L wires were drawn to induce a change from paramagnetic austenitic into ferromagnetic martensitic microstructure. The changes in magnetic behavior were quantified by analyzing the geometric distortion in spin echo and the geometric distortion and intravoxel dephasing in gradient echo images at 0.5, 1.5 and 3 T. The results of both imaging strategies were in agreement and in accordance with independent measurements with a vibrating sample magnetometer. Drawing wire to 2% of its cross-sectional area was found to increase the volume fraction of the ferromagnetic martensite from 0.3% to 80% and to enhance the magnetization up to two or three orders of magnitude. The results demonstrate the potential of MRI to locate and quantify stress-induced changes in the magnetic properties of alloys in a completely noninvasive and nondestructive way.