In Vitro Maturation, In Vitro Oogenesis, and Ovarian Longevity.

This paper will review a remarkable new approach to in vitro maturation "IVM" of oocytes from ovarian tissue, based on our results with in vitro oogenesis from somatic cells. As an aside benefit we also have derived a better understanding of ovarian longevity from ovary transplant. We have found that primordial follicle recruitment is triggered by tissue pressure gradients. Increased pressure holds the follicle in meiotic arrest and prevents recruitment. Therefore recruitment occurs first in the least dense inner tissue of the cortico-medullary junction. Many oocytes can be obtained from human ovarian tissue and mature to metaphase 2 in vitro with no need for ovarian stimulation. Ovarian stimulation may only be necessary for removing the oocyte from the ovary, but this can also be accomplished by simple dissection at the time of ovary tissue cryopreservation. By using surgical dissection of the removed ovary, rather than a needle stick, we can obtain many oocytes from very small follicles not visible with ultrasound. A clearer understanding of ovarian function has come from in vitro oogenesis experiments, and that explains why IVM has now become so simple and robust. Tissue pressure (and just a few "core genes" in the mouse) direct primordial follicle recruitment and development to mature oocyte, and therefore also control ovarian longevity. There are three distinct phases to oocyte development both in vitro and in vivo: in vitro differentiation "IVD" which is not gonadotropin sensitive (the longest phase), in vitro gonadotropin sensitivity "IVG" which is the phase of gonadotropin stimulation to prepare for meiotic competence, and IVM to metaphase II. On any given day 35% of GVs in ovarian tissue have already undergone "IVD" and "IVG" in vivo, and therefore are ready for IVM.

In-vitro maturation and transplantation of cryopreserved ovary tissue: understanding ovarian longevity.

RESEARCH QUESTION: Is it possible to use experience gained from 24 years of frozen ovarian transplantation, and from recent experience with in-vitro gametogenesis to accomplish simple and robust in-vitro maturation (IVM) of oocytes from human ovarian tissue? DESIGN: A total of 119 female patients between age 2 and 35 years old underwent ovary cryopreservation (as well as in-vitro maturation of oocytes and IVM in the last 13 individuals) over a 24-year period. Up to 22 years later, 17 returned to have their ovary tissue thawed and transplanted back. RESULTS: Every woman had a return of ovarian function 5 months after transplant, similar to previous observations. As observed before, anti-Müllerian hormone (AMH) concentration rose as FSH fell 4 months later. The grafts continued to work up to 8 years. Of the 17, 13 (76%) became pregnant with intercourse at least once, resulting in 19 healthy live births, including six live births from three women who had had leukaemia. Of the harvested germinal vesicle oocytes, 35% developed with simple culture media into mature metaphase II oocytes. CONCLUSIONS: The authors concluded the following. First, ovary tissue cryopreservation is a robust method for preserving fertility even for women with leukaemia, without a need to delay cancer treatment. Second, many mature oocytes can often be obtained from ovary tissue with simple media and no need for ovarian stimulation. Third, ovarian stimulation only be necessary for removing the oocyte from the ovary, which can also be accomplished by simple dissection at the time of ovary freezing. Finally, pressure and just eight 'core genes' control primordial follicle recruitment and development.

P2X3 receptor upregulation in trigeminal ganglion neurons through TNFα production in macrophages contributes to trigeminal neuropathic pain in rats.

BACKGROUND: Trigeminal neuralgia is a characteristic disease that manifests as orofacial phasic or continuous severe pain triggered by innocuous orofacial stimulation; its mechanisms are not fully understood. In this study, we established a new animal model of trigeminal neuralgia and investigated the role of P2X3 receptor (P2X3R) alteration in the trigeminal ganglion (TG) via tumor necrosis factor alpha (TNFα) signaling in persistent orofacial pain. METHODS: Trigeminal nerve root compression (TNC) was performed in male Sprague-Dawley rats. Changes in the mechanical sensitivity of whisker pad skin, amount of TNFα in the TG, and number of P2X3R and TNF receptor-2 (TNFR2)-positive TG neurons were assessed following TNC. The effects of TNFR2 antagonism in TG and subcutaneous P2X3R antagonism on mechanical hypersensitivity following TNC were examined. RESULTS: TNC induced unilateral continuous orofacial mechanical allodynia, which was depressed by carbamazepine. The accumulation of macrophages showing amoeboid-like morphological changes and expression of TNFα in the TG was remarkably increased following TNC treatment. The number of P2X3R- and TNFR2-positive TG neurons innervating the orofacial skin was significantly increased following TNC. TNFα was released from activated macrophages that occurred in the TG following TNC, and TNFR2 antagonism in the TG significantly diminished the TNC-induced increase in P2X3R-immunoreactive TG neurons. Moreover, subcutaneous P2X3R antagonism in the whisker pad skin significantly depressed TNC-induced mechanical allodynia. CONCLUSIONS: Therefore, it can be concluded that the signaling of TNFα released from activated macrophages in the TG induces the upregulation of P2X3R expression in TG neurons innervating the orofacial region, resulting in orofacial mechanical allodynia following TNC.

Pigmented fungiform papillae of the tongue in a Japanese child.

Pigmented fungiform papillae of the tongue (PFPT) does not require invasive investigation and treatment. However, if the patient requests treatment for aesthetic reasons, and the pigmentation is focally distributed, an excisional biopsy can be chosen for both diagnosis and treatment.

Mechanical stress accompanied with nuclear rotation is involved in the dormant state of mouse oocytes.

The most immature oocytes remain dormant in primordial follicles in the ovary, ensuring the longevity of female reproductive life. Despite its biological and clinical importance, knowledge of mechanisms regulating the dormant state remains limited. Here, we show that mechanical stress plays a key role in maintaining the dormant state of the oocytes in primordial follicles in mice. Transcriptional and histological analyses revealed that oocytes were compressed by surrounding granulosa cells with extracellular matrix. This environmental state is functionally crucial, as oocytes became activated upon loosening the structure and the dormancy was restored by additional compression with exogenous pressure. The nuclei of oocytes in primordial follicles rotated in response to the mechanical stress. Pausing the rotation triggered activation of oocytes through nuclear export of forkhead box O3 (FOXO3). These results provide insights into the mechanisms by which oocytes are kept dormant to sustain female reproductive life.

Hypoxia induces the dormant state in oocytes through expression of Foxo3.

In mammals, most immature oocytes remain dormant in the primordial follicles to ensure the longevity of female reproductive life. A precise understanding of mechanisms underlying the dormancy is important for reproductive biology and medicine. In this study, by comparing mouse oogenesis in vivo and in vitro, the latter of which bypasses the primordial follicle stage, we defined the gene-expression profile representing the dormant state of oocytes. Overexpression of constitutively active FOXO3 partially reproduced the dormant state in vitro. Based on further gene-expression analysis, we found that a hypoxic condition efficiently induced the dormant state in vitro. The effect of hypoxia was severely diminished by disruption of the Foxo3 gene and inhibition of hypoxia-inducible factors. Our findings provide insights into the importance of environmental conditions and their effectors for establishing the dormant state.

Stem Cell-Derived Human Gametes: The Public Engagement Imperative.

The implications of scientific breakthroughs are rarely faced up to in advance of their realization. Stem cell-derived human gametes, a disruptive technology in waiting, are likely to recapitulate this historic pattern absent active intervention. Herein we call for the conduct of thoughtful ante hoc deliberations on the prospect of stem cell-derived human gametes with an eye toward minimizing potential untoward post hoc regulatory or statutory impositions.

Detection of Bidirectional Promoter-Derived lncRNAs from Small-Scale Samples Using Pre-Amplification-Free Directional RNA-seq Method.

Development of high-throughput sequencing technologies has uncovered the immensity of the long noncoding RNA (lncRNA) world. Divergently transcribed lncRNAs from bidirectional gene promoters, called promoter-associated noncoding RNAs (pancRNAs), account for ~20% of the total number of lncRNAs, and this major fraction is involved in many biological processes, such as development and cancer formation. Recently, we have found that the pancRNAs activate their partner genes, as represented by the fact that pancIl17d, a pancRNA that is transcribed from the antisense strand of the promoter region of Interleukin 17d (Il17d) at the onset of zygotic gene activation (ZGA), is essential for mouse preimplantation development through Il17d upregulation. The discovery of the expression of a specific set of pancRNAs during ZGA was achieved by using a method that generates directional RNA-seq libraries from small-scale samples. Although there are several methods available for small-scale samples, most of them require a pre-amplification procedure that frequently generates some amplification biases toward a subset of transcripts. We provide here a highly sensitive and reproducible method based on the preparation of directional RNA-seq libraries from as little as 100 mouse oocytes or embryos without pre-amplification for the quantification of lncRNAs as well as mRNAs.

The Role of Nerve Growth Factor (NGF) and Its Precursor Forms in Oral Wound Healing.

Nerve growth factor (NGF) and its different precursor forms are secreted into human saliva by salivary glands and are also produced by an array of cells in the tissues of the oral cavity. The major forms of NGF in human saliva are forms of pro-nerve growth factor (pro-NGF) and not mature NGF. The NGF receptors tropomyosin-related kinase A (TrkA) and p75 neurotrophin receptor (p75NTR) are widely expressed on cells in the soft tissues of the human oral cavity, including keratinocytes, endothelial cells, fibroblasts and leukocytes, and in ductal and acinar cells of all types of salivary glands. In vitro models show that NGF can contribute at most stages in the oral wound healing process: restitution, cell survival, apoptosis, cellular proliferation, inflammation, angiogenesis and tissue remodeling. NGF may therefore take part in the effective wound healing in the oral cavity that occurs with little scarring. As pro-NGF forms appear to be the major form of NGF in human saliva, efforts should be made to study its function, specifically in the process of wound healing. In addition, animal and clinical studies should be initiated to examine if topical application of pro-NGF or NGF can be a therapy for chronic oral ulcerations and wounds.

Complete in vitro generation of fertile oocytes from mouse primordial germ cells.

Reconstituting gametogenesis in vitro is a key goal for reproductive biology and regenerative medicine. Successful in vitro reconstitution of primordial germ cells and spermatogenesis has recently had a significant effect in the field. However, recapitulation of oogenesis in vitro remains unachieved. Here we demonstrate the first reconstitution, to our knowledge, of the entire process of mammalian oogenesis in vitro from primordial germ cells, using an estrogen-receptor antagonist that promotes normal follicle formation, which in turn is crucial for supporting oocyte growth. The fundamental events in oogenesis (i.e., meiosis, oocyte growth, and genomic imprinting) were reproduced in the culture system. The most rigorous evidence of the recapitulation of oogenesis was the birth of fertile offspring, with a maximum of seven pups obtained from a cultured gonad. Moreover, cryopreserved gonads yielded functional oocytes and offspring in this culture system. Thus, our in vitro system will enable both innovative approaches for a deeper understanding of oogenesis and a new avenue to create and preserve female germ cells.

Simultaneous administration of recombinant tissue plasminogen activator and edaravone in acute cerebral ischemic stroke patients.

Among the 1052 patients admitted to our hospital because of cerebral infarction between January 1, 2007, and December 31, 2010, we report the treatment outcomes of 48 patients (4.6% of all patients) who received recombinant tissue plasminogen activator (rt-PA) therapy (simultaneously combined with edaravone) within 3 hours after the onset of infarction. Twenty (41.7%) patients started receiving edaravone before rt-PA administration, and 28 patients (58.3%) started receiving rt-PA and edaravone simultaneously. The patients had an average age of 73.5 years (range, 55-93 years; male:female, 32:16). Medical histories included hypertension, diabetes mellitus, dyslipidemia, arterial fibrillation, and a smoking history in 23 (47.8%), 7 (14.6%), 8 (16.7%), 29 (60.4%), and 8 (16.7%) of patients, respectively. Regarding the treatment outcome of the therapy, the National Institutes of Health Stroke Scale score, which was 15 points before rt-PA administration, showed a statistically significant improvement to 8 points after rt-PA administration (P < .001). The modified Rankin Scale scores at 90 days after treatment were as follows: 0 in 12 patients (25.0%), 1 in 11 patients (22.9%), 2 in 7 patients (14.6%), 3 in 5 patients (10.4%), 4 in 6 patients (12.5%), 5 in 5 patients (10.4%), and 6 in 2 patients (4.2%). The occluded blood vessel reopened completely in 30 patients (62.5%) and partially in 5 patients (10.4%). Asymptomatic hemorrhage over the entire brain developed in 2 patients (4.2%). Thus, rt-PA therapy in combination with edaravone improved the recanalization rate, reduced the incidence of intracranial hemorrhage, and improved functional prognosis.

[Effect of an oral anti-estrogen agent (mepitiostane) on the regression of intracranial meningiomas in the elderly].

We report here the regression of meningioma following treatment with the anti-estrogen agent mepitiostane in a series of cases. The first case was that of a 72-year-old woman who presented with coma status due to non-communicating hydrocephalus. A large presumed meningioma within the cerebello-pontine angle was detected on gadolinium-enhanced magnetic resonance imaging (MRI). The patient recovered from the neurological deficit following endoscopic third ventriculostomy treatment, and was administered mepitiostane (10mg/day) orally. Gadolinium-enhanced MRI showed a marked regression (85%) of the meningioma following 60 months of oral medication. The second case was that of a 79-year-old woman with no neurological deficit; however, a presumed meningioma located in the frontal skull base was detected on gadolinium-enhanced MRI. Mepitiostane (10mg/day) was administered orally. Again, a marked regression (88%) of the meningioma was demonstrated after 115 months of oral medication. The third case was that of a 71-year-old woman who presented with right visual disturbance and a visual field defect. Gadolinium-enhanced MRI demonstrated a presumed meningioma located in the left sphenoidal bone. Mepitiostane (20mg/day) was administered orally. An 79% regression of the meningioma was observed after 21 months of oral medication. In these three cases, the marked reduction in meningioma following anti-estrogen agent (mepitiostane) administration suggested that this oral medication could be an effective therapeutic option in elderly patients.

Induction of primordial germ cell-like cells from mouse embryonic stem cells by ERK signal inhibition.

Primordial germ cells (PGCs) are embryonic germ cell precursors. Specification of PGCs occurs under the influence of mesodermal induction signaling during in vivo gastrulation. Although bone morphogenetic proteins and Wnt signaling play pivotal roles in both mesodermal and PGC specification, the signal regulating PGC specification remains unknown. Coculture of mouse embryonic stem cells (ESCs) with OP9 feeder cells induces mesodermal differentiation in vitro. Using this mesodermal differentiation system, we demonstrated that PGC-like cells were efficiently induced from mouse ESCs by extracellular signal-regulated kinase (ERK) signaling inhibition. Inhibition of ERK signaling by a MAPK/ERK kinase (MEK) inhibitor upregulated germ cell marker genes but downregulated mesodermal genes. In addition, the PGC-like cells showed downregulation of DNA methylation and formed pluripotent stem cell colonies upon treatment with retinoic acid. These results show that inhibition of ERK signaling suppresses mesodermal differentiation but activates germline differentiation program in this mesodermal differentiation system. Our findings provide a new insight into the signaling networks regulating PGC specification.

Perspectives of germ cell development in vitro in mammals.

Pluripotent stem cells, such as embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) are able to differentiate into all cell lineages of the embryo proper, including germ cells. This pluripotent property has a huge impact on the fields of regenerative medicine, developmental biology and reproductive engineering. Establishing the germ cell lineage from ESCs/iPSCs is the key biological subject, since it would contribute not only to dissection of the biological processes of germ cell development but also to production of unlimited numbers of functional gametes in vitro. Toward this goal, we recently established a culture system that induces functional mouse primordial germ cells (PGCs), precursors of all germ cells, from mouse ESCs/iPSCs. The successful in vitro production of PGCs arose from the study of pluripotent cell state, the signals inducing PGCs and the technology of transplantation. However, there are many obstacles to be overcome for the robust generation of mature gametes or for application of the culture system to other species, including humans and livestock. In this review, we discuss the requirements for a culture system to generate the germ cell lineage from ESCs/iPSCs.

Generation of eggs from mouse embryonic stem cells and induced pluripotent stem cells.

Oogenesis is an integrated process through which an egg acquires the potential for totipotency, a fundamental condition for creating new individuals. Reconstitution of oogenesis in a culture that generates eggs with proper function from pluripotent stem cells (PSCs) is therefore one of the key goals in basic biology as well as in reproductive medicine. Here we describe a stepwise protocol for the generation of eggs from mouse PSCs, such as embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). ESCs and iPSCs are first induced into primordial germ cell-like cells (PGCLCs) that are in turn aggregated with somatic cells of female embryonic gonads, the precursors for adult ovaries. Induction of PGCLCs followed by aggregation with the somatic cells takes up to 8 d. The aggregations are then transplanted under the ovarian bursa, in which PGCLCs grow into germinal vesicle (GV) oocytes in ∼1 month. The PGCLC-derived GV oocytes can be matured into eggs in 1 d by in vitro maturation (IVM), and they can be fertilized with spermatozoa by in vitro fertilization (IVF) to obtain healthy and fertile offspring. This method provides an initial step toward reconstitution of the entire process of oogenesis in vitro.

Distribution of nerve growth factor, pro-nerve growth factor, and their receptors in human salivary glands.

Nerve growth factor (NGF) is a pluripotent mediator that is present in a range of human tissues. Nerve growth factor was originally considered important only in neuronal homeostasis and pathophysiology, but later it was also implicated in the pathophysiology of inflammation, epithelial differentiation, and wound healing. In this study, the distribution of nerve growth factor beta (NGF-ß) and pro-NGF, and their receptors - tyrosine kinase A (TrkA) and p75(NTR) - was examined in human parotid, submandibular, sublingual, and labial salivary glands by immunohistochemistry. Intercalated, striated, and collecting-ducts in all gland types showed strong staining for pro-NGF but only weak cytoplasmic or sparse nuclear staining for NGF-ß. Tyrosine kinase A was strongly expressed in the ducts of all gland types, whereas p75(NTR) expression was mainly confined to collecting ducts. In acini, no or only weak cytoplasmic staining was found for all markers, and some nuclei stained positive for NGF-ß, pro-NGF, and TrkA. Western blotting of saliva showed secretion of several forms of pro-NGF, while no mature NGF-ß was detected. Salivary pro-NGF may play a role in oral wound healing.

Offspring from oocytes derived from in vitro primordial germ cell-like cells in mice.

Reconstitution of female germ cell development in vitro is a key challenge in reproductive biology and medicine. We show here that female (XX) embryonic stem cells and induced pluripotent stem cells in mice are induced into primordial germ cell-like cells (PGCLCs), which, when aggregated with female gonadal somatic cells as reconstituted ovaries, undergo X-reactivation, imprint erasure, and cyst formation, and exhibit meiotic potential. Upon transplantation under mouse ovarian bursa, PGCLCs in the reconstituted ovaries mature into germinal vesicle-stage oocytes, which then contribute to fertile offspring after in vitro maturation and fertilization. Our culture system serves as a robust foundation for the investigation of key properties of female germ cells, including the acquisition of totipotency, and for the reconstitution of whole female germ cell development in vitro.

Reconstitution of the mouse germ cell specification pathway in culture by pluripotent stem cells.

The generation of properly functioning gametes in vitro requires reconstitution of the multistepped pathway of germ cell development. We demonstrate here the generation of primordial germ cell-like cells (PGCLCs) in mice with robust capacity for spermatogenesis. PGCLCs were generated from embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) through epiblast-like cells (EpiLCs), a cellular state highly similar to pregastrulating epiblasts but distinct from epiblast stem cells (EpiSCs). Reflecting epiblast development, EpiLC induction from ESCs/iPSCs is a progressive process, and EpiLCs highly competent for the PGC fate are a transient entity. The global transcription profiles, epigenetic reprogramming, and cellular dynamics during PGCLC induction from EpiLCs meticulously capture those associated with PGC specification from the epiblasts. Furthermore, we identify Integrin-ß3 and SSEA1 as markers that allow the isolation of PGCLCs with spermatogenic capacity from tumorigenic undifferentiated cells. Our findings provide a paradigm for the first step of in vitro gametogenesis.

Genome-wide identification of targets and function of individual MicroRNAs in mouse embryonic stem cells.

Mouse Embryonic Stem (ES) cells express a unique set of microRNAs (miRNAs), the miR-290-295 cluster. To elucidate the role of these miRNAs and how they integrate into the ES cell regulatory network requires identification of their direct regulatory targets. The difficulty, however, arises from the limited complementarity of metazoan miRNAs to their targets, with the interaction requiring as few as six nucleotides of the miRNA seed sequence. To identify miR-294 targets, we used Dicer1-null ES cells, which lack all endogenous mature miRNAs, and introduced just miR-294 into these ES cells. We then employed two approaches to discover miR-294 targets in mouse ES cells: transcriptome profiling using microarrays and a biochemical approach to isolate mRNA targets associated with the Argonaute2 (Ago2) protein of the RISC (RNA Induced Silencing Complex) effector, followed by RNA-sequencing. In the absence of Dicer1, the RISC complexes are largely devoid of mature miRNAs and should therefore contain only transfected miR-294 and its base-paired targets. Our data suggest that miR-294 may promote pluripotency by regulating a subset of c-Myc target genes and upregulating pluripotency-associated genes such as Lin28.

Expression of the T-cell-specific adapter protein in oral epithelium.

The multifunctional T-cell-specific adapter protein (TSAd) was originally described in T cells but is also expressed in epithelial cells from the respiratory tract and in endothelium. In this study, we found expression of TSAd messenger RNA (mRNA) and protein in both human and murine oral mucosal epithelium as well as in human primary oral keratinocyte cell cultures. In TSAd(-/-) mice, the mucosa and skin appeared macroscopically normal, but severe disturbances were observed in the fine structures of the basal membrane and intercellular epithelial spaces upon analysis using transmission electron microscopy. Oral epithelial cells from TSAd(-/-) mice displayed decreased migration compared with cells from wild-type mice, whereas overexpression of TSAd in a human epithelial cell line resulted in impaired proliferation. This study is the first to show that TSAd is expressed in normal oral mucosa, that it is important for the normal ultrastructural morphology of the epithelium and the basal membrane, and that it is involved in the migration and proliferation of oral keratinocytes.