miRNA Nomenclature: A View Incorporating Genetic Origins, Biosynthetic Pathways, and Sequence Variants.

High-throughput sequencing of miRNAs has revealed the diversity and variability of mature and functional short noncoding RNAs, including their genomic origins, biogenesis pathways, sequence variability, and newly identified products such as miRNA-offset RNAs (moRs). Here we review known cases of alternative mature miRNA-like RNA fragments and propose a revised definition of miRNAs to encompass this diversity. We then review nomenclature guidelines for miRNAs and propose to extend nomenclature conventions to align with those for protein-coding genes established by international consortia. Finally, we suggest a system to encompass the full complexity of sequence variations (i.e., isomiRs) in the analysis of small RNA sequencing experiments.

Neuroimaging correlates of everyday action in dementia.

The everyday, functional impairments associated with dementia remain poorly understood from a neuropsychological perspective. This study investigated relations between brain structure volumes and two measures of everyday action-caregiver questionnaire and direct assessment-in 57 participants with dementia. Results showed that caregiver ratings reflecting more functional impairment were strongly associated with smaller volumes of deep white matter. Direct assessment of everyday task performance in a subsample revealed relations between unique neurological substrates and discrete everyday action error types. Findings emphasize differences in functional assessment methods and highlight the role of white matter in functional deficits in dementia.

The Mouse Tumor Biology Database (MTB): a central electronic resource for locating and integrating mouse tumor pathology data.

The Mouse Tumor Biology Database (MTB) is designed to provide an electronic data storage, search, and analysis system for information on mouse models of human cancer. The MTB includes data on tumor frequency and latency, strain, germ line, and somatic genetics, pathologic notations, and photomicrographs. The MTB collects data from the primary literature, other public databases, and direct submissions from the scientific community. The MTB is a community resource that provides integrated access to mouse tumor data from different scientific research areas and facilitates integration of molecular, genetic, and pathologic data. Current status of MTB, search capabilities, data types, and future enhancements are described in this article.

Effect of in vitro maturation of mouse oocytes on the health and lifespan of adult offspring.

BACKGROUND: In vitro maturation of oocytes can, in some circumstances, provide an alternative approach to gonadotrophin-induced maturation in clinical settings. However, the consequences of these protocols on the long-term health of offspring are unknown. Here, the long-term health status and lifespans of offspring produced by in vitro maturation of mouse oocytes was compared with that of oocytes induced to mature in vivo using gonadotrophin treatment. METHODS: Mouse oocytes were matured in vitro using both an established optimized system and in the absence of amino acids to produce a suboptimal condition for maturation. Oocytes induced to mature in vivo with gonadotrophins constituted the control group. All metaphase II oocytes were fertilized in vitro and transferred at the 2-cell stage to the oviducts of pseudo-pregnant foster mothers for development to term. Offspring were subjected to a wide variety of physiological and behavioral tests for the first year of life and natural lifespan determined. RESULTS: There was no difference among the groups in lifespan or in most of the physiological and behavioral analyses. However, the pulse rate and cardiac output were slightly, but significantly, reduced in the optimized in vitro matured group compared with the in vivo matured group (P = 0.0119 and P = 0.0197, respectively). Surprisingly, these decreases were largely abrogated in the in vitro group matured without amino acids. CONCLUSIONS: Evidence presented here using a mouse model suggests that the in vitro maturation of oocytes has minimal effects on the long-term health of offspring. However, a finding of slight reductions in pulse rate and cardiac output may focus future clinical attention.

Oocytes are required for the preantral granulosa cell to cumulus cell transition in mice.

Preantral granulosa cells (PAGCs) differentiate into cumulus cells following antrum formation. Cumulus cells, but not PAGCs, are competent to undergo expansion. Experiments reported here tested the respective roles of both oocytes and FSH in the transition of preantral granulosa cells to cumulus cells competent to undergo expansion. PAGC-oocyte complexes were cultured with or without a low dose of FSH (0.005 IU/ml) and isolated PAGCs were cultured with or without oocytes. At the end of culture, complexes or isolated PAGCs were tested for their ability to undergo cumulus expansion and upregulate expansion transcripts in response to EGF or FSH (0.5 IU/ml). The ability to undergo expansion in response to EGF required the presence of oocytes but not FSH during the culture period. Likewise, complexes isolated from the ovaries of hypogonadal mice, which lack circulating gonadotropins, underwent expansion in response to EGF, but not FSH. In contrast, the ability to activate MAPK3/1 and MAPK14 and undergo expansion in response to FSH required prior exposure to low doses of FSH. However, these low levels (0.005 or 0.025 IU FSH/ml) suppressed expression of Slc38a3 and Amh, two transcripts highly expressed in cumulus cells, suggesting opposing effects of FSH on cumulus cell differentiation. In conclusion, the ability to undergo expansion in response to FSH requires prior exposure to FSH during development, while oocyte-derived factors alone are sufficient to promote the ability to undergo expansion in response to EGF. These results highlight the crucial role of oocytes in driving the differentiation of PAGCs into cumulus cells during the preantral to antral follicle transition.

The preantral granulosa cell to cumulus cell transition in the mouse ovary: development of competence to undergo expansion.

The transition of preantral to antral follicles is one of the major steps in follicular development, yet little is known about the molecular and functional changes that occur as preantral granulosa cells differentiate into cumulus cells. The cumulus oophorus of large antral follicles undergoes expansion in response to the preovulatory surge of gonadotropins, but preantral granulosa cells do not. The objective of this project was to determine the molecular mechanisms underlying this differential response. Cumulus expansion in vitro requires secretion of cumulus-expansion enabling factors (CEEFs) by the oocyte and stimulation by a ligand, epidermal growth factor (EGF) or follicle-stimulating hormone (FSH). This combined stimulation results in activation of MAPKs (MAPK3/1 (formerly ERK1/2) and MAPK14 (formerly p38)) and increased Has2, Ptgs2, Tnfaip6 and Ptx3 mRNA levels, all of which are required for cumulus expansion. Only fully-grown oocytes from antral follicles were competent to enable expansion and increases in expansion-related transcripts in cumulus cells, whereas growing oocytes of preantral follicles did not. To assess the competence of preantral granulosa cells to generate responses associated with expansion, they were treated with FSH or EGF and co-cultured with fully-grown oocytes secreting CEEFs. MAPKs were activated by EGF in preantral granulosa cells to essentially the same levels as in cumulus cells. Preantral granulosa cells treated with EGF, but not those treated with FSH increased Has2, Ptgs2 and Ptx3 mRNAs to 17-96% of the levels observed in cumulus cells. In contrast, the level of Tnfaip6 mRNA was minimally stimulated in preantral granulosa cells. Therefore, preantral granulosa cells do not undergo expansion for two fundamental reasons. First, the growing oocytes of preantral follicles do not secrete active CEEFs. Second, activation of MAPKs alone in preantral granulosa cells, even in the presence of CEEFs, is not sufficient to increase the expression of essential transcripts, particularly Tnfaip6 mRNA. Thus, preantral granulosa cells differ from cumulus cells in CEEF-dependent processes downstream of the activation of MAPKs.

A synthetic analogue of meiosis-activating sterol (FF-MAS) is a potent agonist promoting meiotic maturation and preimplantation development of mouse oocytes maturing in vitro.

BACKGROUND: Follicular fluid-meiosis-activating sterol (FF-MAS) is a factor present in the pre-ovulatory follicle during the time of oocyte maturation. In mouse oocytes maturing in vitro, FF-MAS promotes the completion of meiotic maturation to metaphase II (MII) and improves competence to complete the 2-cell stage to blastocyst transition. We produced analogues of FF-MAS and selected three on the basis of potency to promote the resumption of meiosis by mouse oocytes maintained in meiotic arrest by hypoxanthine. The objective of this study was to determine whether these FF-MAS analogues also affect the quality of oocytes maturing in vitro with respect to the completion of meiotic maturation and augmenting the frequency of development to the blastocyst stage after fertilization in vitro. METHODS: Cumulus cell-enclosed oocytes were isolated from the small antral follicles of 18 or 20 day post-natal mice. These oocytes normally have a reduced competence to complete meiotic maturation and preimplantation embryo development. Oocytes were isolated at the germinal vesicle stage and matured in vitro using media supplemented with 0.1% ethanol, 1 micromol/l FF-MAS, or 0.1-10 micromol/l FF-MAS analogues ZK255884 (884), ZK255933 (933) and ZK255991 (991). Oocytes that progressed to MII were fertilized in vitro and the percentage developing to the 2-cell and blastocyst stages was determined. RESULTS: At 1 micromol/l, 991 and 933 increased the portion of oocytes progressing to MII, whereas the lowest dose of 991 and 884 was ineffective. Treatment of maturing oocytes with either 0.1 or 1 micromol/l 933 dramatically increased oocyte competence to complete preimplantation development. CONCLUSIONS: The synthetic analogue of FF-MAS, ZK255933, is a potent agonist that improves the quality of mouse oocytes matured in vitro. This compound may therefore have therapeutic value for treatment of oocytes from women undergoing therapy for infertility owing to poor oocyte quality.

New mouse genetic models for human contraceptive development.

Genetic strategies for the post-genomic sequence age will be designed to provide information about gene function in a myriad of physiological processes. Here an ENU mutagenesis program (http://reprogenomics.jax.org) is described that is generating a large resource of mutant mouse models of infertility; male and female mutants with defects in a wide range of reproductive processes are being recovered. Identification of the genes responsible for these defects, and the pathways in which these genes function, will advance the fields of reproduction research and medicine. Importantly, this program has potential to reveal novel human contraceptive targets.

The Gene Ontology (GO) database and informatics resource.

The Gene Ontology (GO) project (http://www. geneontology.org/) provides structured, controlled vocabularies and classifications that cover several domains of molecular and cellular biology and are freely available for community use in the annotation of genes, gene products and sequences. Many model organism databases and genome annotation groups use the GO and contribute their annotation sets to the GO resource. The GO database integrates the vocabularies and contributed annotations and provides full access to this information in several formats. Members of the GO Consortium continually work collectively, involving outside experts as needed, to expand and update the GO vocabularies. The GO Web resource also provides access to extensive documentation about the GO project and links to applications that use GO data for functional analyses.

Oocyte control of ovarian follicular development and function in mammals.

A new perspective on ovarian follicular development has emerged over the last decade. Whereas the oocyte was previously considered only a passive recipient of developmental signals from oocyte-associated granulosa cells, it is now clear that communication between oocytes and granulosa cells is bidirectional. A complex interplay of regulatory factors governs the development of both types of cell. This interplay is essential not only for oocyte development but also for follicular development, beginning with the initial assembly of the primordial follicle and continuing throughout ovulation. The existence of an oocyte-granulosa cell regulatory loop, essential for normal follicular differentiation as well as for the production of an oocyte competent to undergo fertilization and embryogenesis, is proposed. Although gonadotrophins are essential for driving the differentiation of granulosa cell phenotypes, within its sphere of influence, the oocyte is probably the dominant factor determining the direction of differentiation and the function of the granulosa cells associated with it.

Oosp1 encodes a novel mouse oocyte-secreted protein.

Oocyte-somatic cell communication is necessary for normal ovarian function. However, the identities of the majority of oocyte-secreted proteins remain unknown. A novel cDNA encoding mouse oocyte-secreted protein 1 (OOSP1) was identified using a modified subtractive hybridization screen. The Oosp1 cDNA encodes a 202-amino acid protein that contains a 21-amino acid signal peptide sequence, 5 putative N-linked glycosylation consensus sequences, and 6 cysteines that are predicted to form 3 disulfide bonds. OOSP1 shares amino acid identity with placental-specific protein 1 (PLAC1), a secreted protein expressed in the placenta and the ectoplacental cone. The Oosp1 mRNA is approximately 1.0 kb and is present at high levels in the oocytes of adult ovaries and at lower levels in the spleen. The mouse Oosp1 gene is 5 exons, spans greater than 16.4 kb, and localizes to chromosome 19 at a position that shares synteny with human chromosome 11q12-11q13. The identification of OOSP1 as a new oocyte-secreted protein permits future in vitro and in vivo functional analyses to define its role in ovarian folliculogenesis.

Availability and characterization of transgenic and knockout mice with behavioral manifestations: where to look and what to search for.

Mice altered by transgenesis or gene targeting ("knockouts") have increasingly been employed as alternative effective tools in elucidating the genetic basis of neurophysiology and behavior. Standardization of specific behavioral paradigms and phenotyping strategies will ensure that these behavioral mouse mutants offer robust models for evaluating the efficacy of novel therapeutics in the treatment of hereditary neurological disorders. The Induced Mutant Resource (IMR) at The Jackson Laboratory (Bar Harbor, Maine, USA) imports, cryopreserves, develops, maintains, and distributes to the research community biomedically valuable stocks of transgenic and targeted mutant mice. Information on behavioral and neurological strains-including a phenotypic synopsis, husbandry requirements, strain availability, and genetic typing protocols-is available through the IMR database (http://www.jax.org/resources/documents/imr/). A current catalog of available strains is readily accessible via the JAX Mice Web site at http://jaxmice.jax.org/index.shtml. In addition, The Jackson Laboratory is now home to TBASE (http://tbase.jax.org/), a comprehensive, community database whose primary focus is on mouse knockouts. TBASE accommodates an exhaustive bibliographical resource for transgenic and knockout mice and provides a detailed phenotypic characterization of numerous behavioral knockouts that is primarily extracted from the literature. Concerted efforts to merge the two resources into a new, schematically reformed database are underway.

Evidence that protein kinase C (PKC) participates in the meiosis I to meiosis II transition in mouse oocytes.

Oocytes from LTXBO mice exhibit a delayed entry into anaphase I and frequently enter interphase after the first meiotic division. This unique oocyte model was used to test the hypothesis that protein kinase C (PKC) may regulate the meiosis I-to-meiosis II transition. PKC activity was detected in LTXBO oocytes at prophase I and increased with meiotic maturation, with the highest (P < 0.05) activity observed at late metaphase I (MI). Treatment of late MI-stage oocytes with the PKC inhibitor, bisindolylmaleimide I (BIM), transiently reduced (P < 0.05) M-phase-promoting factor (MPF) activity and promoted (P < 0.05) progression to metaphase II (MII), while mitogen-activated protein kinase (MAPK) activity remained elevated during the MI-to-MII transition. Confocal microscopy analysis of LTXBO oocytes during this transition showed PKC-delta associated with the meiotic spindle and then with the chromosomes at MII. Inhibition of PKC activity also prevented untimely entry into interphase, but only when PKC activity was reduced in oocytes before the progression to MII and thus indicates that the transition into interphase is directly associated with the delayed triggering of anaphase I. Moreover, the defect(s) that initiate activation occur upstream of MAPK, as suppression of PKC activity failed to prevent activation by Mos(tm1Ev)/ Mos(tm1Ev) LTXBO oocytes expressing no detectable MAPK activity. In summary, PKC participates in the regulatory mechanisms that delay entry into anaphase I in LTXBO oocytes, and the disruption promotes untimely entry into interphase. Thus, loss of regulatory control over PKC activity during oocyte maturation disrupts the critical MI-to-MII transition, leading to a precocious exit from meiosis.

Regulation of prostaglandin-endoperoxide synthase 2 messenger ribonucleic acid expression in mouse granulosa cells during ovulation.

Normal ovulation in mice requires PG-endoperoxide synthase 2 (cyclooxygenase-2; COX-2) expression. This study examined the role of the oocyte and other factors in regulating steady state levels of COX-2 messenger RNA (mRNA) in granulosa cells. Multiphasic changes in the expression pattern of COX-2 mRNA were found, with peaks of expression 4 and 12 h after hCG treatment. Changes in relative expression levels in cumulus cells and mural granulosa cells occurred over time, with similar mRNA levels at 4 h, but higher levels in cumulus cells compared with mural granulosa cells at 8 and 12 h post-hCG. In cultured mural granulosa cells, LH, FSH, and oocytes promoted COX-2 mRNA expression concurrent with the first expression peak in vivo. At the same time, FSH, but not LH, treatment of cultured cumulus-oocyte complexes (COC) promoted COX-2 mRNA expression in cumulus cells. This response of cumulus cells to FSH treatment was largely dependent on the presence of either fully grown germinal vesicle stage or maturing oocytes, but not growing oocytes. At 8 h, COX-2 mRNA expression in FSH-stimulated COC was lower than at 4 h; however, oocyte coculture promoted COX-2 mRNA expression in cumulus cells. No second peak in expression occurred in cultured COC. However, coculture of COC with follicle walls promoted COX-2 mRNA expression in cumulus cells 12 h post-hCG; an effect augmented by oocytes. Therefore, the oocyte resident within ovulatory follicles produces a factor(s) that promotes expression of COX-2 mRNA by cumulus cells and possibly by mural granulosa cells. Thus, the oocyte probably plays an important role in promoting ovulation. However, the multiphasic changes in the pattern of COX-2 expression appear orchestrated by non-oocyte-derived factors.

Intercellular communication via connexin43 gap junctions is required for ovarian folliculogenesis in the mouse.

The ovarian follicle in mammals is a functional syncytium, with the oocyte being coupled with the surrounding cumulus granulosa cells, and the cumulus cells being coupled with each other and with the mural granulosa cells, via gap junctions. The gap junctions coupling granulosa cells in mature follicles contain several different connexins (gap junction channel proteins), including connexins 32, 43, and 45. Connexin43 immunoreactivity can be detected from the onset of folliculogenesis just after birth and persists through ovulation. In order to assess the importance of connexin43 gap junctions for postnatal folliculogenesis, we grafted ovaries from late gestation mouse fetuses or newborn pups lacking connexin43 (Gja1(-)/Gja1(-)) into the kidney capsules of adult females and allowed them to develop for up to 3 weeks (this was necessitated by the neonatal lethality caused by the mutation). By the end of the graft period, tertiary (antral) follicles had developed in grafted normal (wild-type or heterozygote) ovaries. Most follicles in Gja1(-)/Gja1(-) ovaries, however, failed to become multilaminar, with the severity of the effect depending on strain background. Dye transfer experiments indicated that intercellular coupling between granulosa cells is reduced, but not abolished, in the absence of connexin43, consistent with the presence of additional connexins. These results suggest that coupling between granulosa cells mediated specifically by connexin43 channels is required for continued follicular growth. Measurements of oocyte diameters revealed that oocyte growth in mutant follicles is retarded, but not arrested, despite the arrest of folliculogenesis. The mutant follicles are morphologically abnormal: the zona pellucida is poorly developed, the cytoplasm of both granulosa cells and oocytes is vacuolated, and cortical granules are absent from the oocytes. Correspondingly, the mutant oocytes obtained from 3-week grafts failed to undergo meiotic maturation and could not be fertilized, although half of the wild-type oocytes from 3-week grafted ovaries could be fertilized. We conclude that connexin43-containing gap junction channels are required for expansion of the granulosa cell population during the early stages of follicular development and that failure of the granulosa cell layers to develop properly has severe consequences for the oocyte.

Defective zonae pellucidae in Zp2-null mice disrupt folliculogenesis, fertility and development.

All vertebrate eggs are surrounded by an extracellular matrix. This matrix is known as the zona pellucida in mammals and is critically important for the survival of growing oocytes, successful fertilization and the passage of early embryos through the oviduct. The mouse zona pellucida is composed of three glycoproteins (ZP1, ZP2 and ZP3), each encoded by a single copy gene. Using targeted mutagenesis in embryonic stem cells, Zp2-null mouse lines have been established. ZP1 and ZP3 proteins continue to be synthesized and form a thin zona matrix in early follicles that is not sustained in pre-ovulatory follicles. The abnormal zona matrix does not affect initial folliculogenesis, but there is a significant decrease in the number of antral stage follicles in ovaries isolated from mice lacking a zona pellucida. Few eggs are detected in the oviduct after stimulation with gonadotropins, and no two-cell embryos are recovered after mating Zp2-null females with normal male mice. The structural defect is more severe than that observed in Zp1-null mice, which have decreased fecundity, but not quite as severe as that observed in Zp3-null mice, which never form a visible zona pellucida and are sterile. Although zona-free oocytes matured and fertilized in vitro can progress to the blastocyst stage, the developmental potential of blastocysts derived from either Zp2- or Zp3-null eggs appears compromised and, after transfer to foster mothers, live births have not been observed. Thus, in addition to its role in fertilization and protection of early embryos, these data are consistent with the zona pellucida maintaining interactions between granulosa cells and oocytes during folliculogenesis that are critical to maximize developmental competence of oocytes.

Transcriptional activity of the mouse oocyte genome: companion granulosa cells modulate transcription and chromatin remodeling.

Chromatin configuration in the germinal vesicle (GV) undergoes dynamic changes during oocyte growth, yet little is known about the mechanisms regulating chromatin remodeling in mouse oocytes. The hypothesis that companion granulosa cells play a role in modulating chromatin configuration and subsequent transcriptional activity in the oocyte genome was tested. Analysis of transcriptional activity, as determined by Br-UTP incorporation, revealed a similar percentage of transcriptionally active and inactive oocytes present in the large antral follicles of mature females. However, gonadotropin stimulation of follicular development induced an increase in the proportion of transcriptionally inactive oocytes. Interestingly, a similar proportion of stage-matched, oocyte-granulosa cell complexes grown in vitro without gonadotropin stimulation displayed chromatin redistribution around the nucleolus and no transcriptional activity. In contrast, when cultured in the absence of companion granulosa cells, transcriptional activity remained unabated in the majority of denuded GV stage oocytes. Extended prophase arrest in fully grown transcriptionally inactive oocyte-granulosa cell complexes had no effect on the progression of meiosis after in vitro maturation. However, it reduced the competence to complete preimplantation embryo development. These results indicate that chromatin redistribution around the nucleolus is associated with transcriptional repression in the GV of both fully grown in vivo-derived oocytes and cultured oocyte-granulosa cell complexes. Moreover, the results presented here suggest that some aspects of intraovarian control mechanisms were abrogated during culture of oocyte-granulosa cell complexes, resulting in a higher proportion of oocytes with "mature" chromatin. Most importantly, companion granulosa cells played an active role in modulating the transcriptional activity of the oocyte genome.