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Richard M Schultz

Publications and source records attributed to Richard M Schultz.

At least 19 recordsLinked to original sources
PubMed2024

Kick-starting the zygotic genome: licensors, specifiers, and beyond.

Zygotic genome activation (ZGA), the first transcription event following fertilization, kickstarts the embryonic program that takes over the control of early development from the maternal products. How ZGA occurs, especially in mammals, is poorly understood due to the limited amount of research materials. With the rapid development of single-cell and low-input technologies, remarkable progress made in the past decade has unveiled dramatic transitions of the epigenomes, transcriptomes, proteomes, and metabolomes associated with ZGA. Moreover, functional investigations are yielding insights into the key regulators of ZGA, among which two major classes of players are emerging: licensors and specifiers. Licensors would control the permission of transcription and its timing during ZGA. Accumulating evidence suggests that such licensors of ZGA include regulators of the transcription apparatus and nuclear gatekeepers. Specifiers would instruct the activation of specific genes during ZGA. These specifiers include key transcription factors present at this stage, often facilitated by epigenetic regulators. Based on data primarily from mammals but also results from other species, we discuss in this review how recent research sheds light on the molecular regulation of ZGA and its executors, including the licensors and specifiers.

Animals↗
PubMed2006

Basonuclin: a novel mammalian maternal-effect gene.

Basonuclin is a zinc-finger protein found in abundance in oocytes. It qualifies as a maternal-effect gene because the source of pre-implantation embryonic basonuclin is maternal. Using a transgenic-RNAi approach, we knocked down basonuclin specifically in mouse oocytes, which led to female sub-fertility. Basonuclin deficiency in oocytes perturbed both RNA polymerase I- and II-mediated transcription, and oocyte morphology was affected (as evidenced by cytoplasmic and cell surface abnormalities). Some of the affected oocytes, however, could still mature to and arrest at metaphase II, and be ovulated. Nevertheless, fertilized basonuclin-deficient eggs failed to develop beyond the two-cell stage, and this pre-implantation failure accounted for the sub-fertility phenotype. These results suggest that basonuclin is a new member of the mammalian maternal-effect genes and, interestingly, differs from the previously reported mammalian maternal-effect genes in that it also apparently perturbs oogenesis.

Animals↗
PubMed2006

Role of calcium signals in early development.

The mammalian egg appears to transduce the duration, amplitude, and temporal presentation of the increase in the intracellular calcium concentration ([Ca(2+)](i)) upon fertilization. These Ca(2+) parameters have important short-term effects on the initiation and completion of early events of egg activation, as well as much later consequences for the extent of peri-implantation development. Recent studies have begun to shed light on how the egg quantitatively interprets the Ca(2+) signal (e.g., by summation of individual Ca(2+) rises) and the mechanisms by which down-stream Ca(2+) effectors, such as Ca(2+)/Calmodulin (CaM)-dependent protein kinase II (CaMKII), utilize this ionic signal to promote biological events that initiate development.

Animals↗
PubMed2006

Deletion of the DNA/RNA-binding protein MSY2 leads to post-meiotic arrest.

Y-box proteins are a well-characterized family of nucleic acid binding proteins that are expressed from bacteria to human. This review will focus on MSY2, a member of the Y-box gene family that is exclusively expressed in male and female germ cells. MSY2 is the mouse ortholog of FRGY2, the Xenopus germ cell-specific protein and the human germ cell protein, Contrin. MSY2 functions as a co-activator of transcription in male germ cells and plays an important role in the translational repression and storage of both paternal and maternal mRNAs in spermatocytes, spermatids and oocytes. Following gene targeting, matings of heterozygotes produce a normal Mendelian ratio with equal numbers of phenotypically normal males and females. However, males and females lacking Msy2 are infertile. In Msy2-null males, spermatogenesis is disrupted in post-meiotic germ cells with many misshapen and multinucleated spermatids. No spermatozoa are found in the epididymis. The germ cell specificity and the critical functions played by this multifunctional DNA- and RNA-binding protein during spermatogenesis make Contrin, the human ortholog of MSY2, an attractive and novel target for male contraception.

Animals↗
PubMed2006

PKB/AKT is involved in resumption of meiosis in mouse oocytes.

BACKGROUND INFORMATION: In fully grown mouse oocytes, a decrease in cAMP concentration precedes and is linked to CDK1 (cyclin-dependent kinase 1) activation. The molecular mechanism for this coupling, however, is not defined. PKB (protein kinase B, also called AKT) is implicated in CDK1 activation in lower species. During resumption of meiosis in starfish oocytes, MYT1, a negative regulator of CDK1, is phosphorylated by PKB in an inhibitory manner. It can imply that PKB is also involved in CDK1 activation in mammalian oocytes. RESULTS: We monitored activation of PKB and CDK1 during maturation of mouse oocytes. PKB phosphorylation and activation preceded GVBD (germinal vesicle breakdown) in oocytes maturing either in vitro or in vivo. Activation was transient and PKB activity was markedly reduced when virtually all of the oocytes had undergone GVBD. PKB activation was independent of CDK1 activity, because although butyrolactone I prevented CDK1 activation and GVBD, PKB was nevertheless transiently phosphorylated and activated. LY-294002, an inhibitor of phosphoinositide 3-kinase-PKB signalling, suppressed activation of PKB and CDK1 as well as resumption of meiosis. OA (okadaic acid)-sensitive phosphatases are involved in PKB-activity regulation, because OA induced PKB hyperphosphorylation. During resumption of meiosis, PKB phosphorylated on Ser(473) is associated with nuclear membrane and centrosome, whereas PKB phosphorylated on Thr(308) is localized on centrosome only. CONCLUSIONS: The results of the present paper indicate that PKB is involved in CDK1 activation and resumption of meiosis in mouse oocytes. The presence of phosphorylated PKB on centrosome at the time of GVBD suggests its important role for an initial CDK1 activation.

4-Butyrolactone↗
PubMed2005

Transcript profiling during mouse oocyte development and the effect of gonadotropin priming and development in vitro.

The molecular basis for acquisition of meiotic and developmental competence, the two main outcomes of oocyte development and essential for producing an egg capable of being fertilized and supporting development to term, is largely unknown. Using microarrays, we characterized global changes in gene expression in oocytes derived from primordial, primary, secondary, small antral, and large antral follicles and used Expression Analysis Systematic Explorer (EASE) to identify biological and molecular processes that accompany these transitions and likely underpin acquisition of meiotic and developmental competence. The greatest degree of change in gene expression occurs during the primordial to primary follicle transition. Of particular interest is that specific chromosomes display significant changes in their overall transcriptional activity and that in some cases these changes are largely confined to specific regions on these chromosomes. We also examined the transcript profile of oocytes that developed in vitro, as well as following eCG priming. Remarkably, the expression profiles only differed by 4% and 2% from oocytes that developed in vivo when compared to oocytes that developed in vitro from either primordial or secondary follicles, respectively. About 1% of the genes were commonly mis-expressed, and EASE analysis revealed there is an over-representation of genes involved in transcription. Developmental competence of oocytes obtained from eCG-primed mice was substantially improved when compared to oocytes obtained from unprimed mice, and this correlated with decreased expression of genes implicated in basal transcription.

Animals↗
PubMed2005

Cyclin A2-CDK2 regulates embryonic gene activation in 1-cell mouse embryos.

Recruitment of maternal mRNA in mice appears essential for embryonic gene activation (EGA) that is initiated in the 1-cell stage. The identity of which recruited mRNAs is responsible, however, is not known. We report here that recruitment of cyclin A2 mRNA may be critical for EGA. Cyclin A2 protein accumulates in pronuclei between 6 and 12 h after fertilization, the time when EGA is initiated. This cyclin A2 may be generated from maternally recruited cyclin A2 mRNA because its accumulation was inhibited by 3'-deoxyadenosine, which inhibits mRNA polyadenylation. When CDK2 activity or pronuclear accumulation of cyclin A2 was inhibited with CDK2 inhibitors or by microinjected siRNAs, respectively, DNA replication was not inhibited but the increase of transcriptional activity was prevented. In addition, microinjection of recombinant cyclin A2-CDK2 protein increased transcriptional activity. Cyclin A2-CDK2 is activated following egg activation, because an increase in phosphorylation of retinoblastoma protein was observed using antibodies that recognize site-specific phosphorylation catalyzed by this kinase and treatment with a CDK2 inhibitor or microinjection with cyclin A2 siRNAs prevented the increase in retinoblastoma protein phosphorylation. These results suggest that recruitment of maternal cyclin A2 mRNA following egg activation is linked to EGA.

Animals↗
PubMed2005

Absence of non-specific effects of RNA interference triggered by long double-stranded RNA in mouse oocytes.

RNA interference (RNAi) is a conserved eukaryotic mechanism by which double-stranded RNA (dsRNA) triggers the sequence-specific degradation of homologous mRNAs. Recent concerns have arisen in mammalian systems about off-target effects of RNAi, as well as an interferon response. Most mammalian cells respond to long dsRNAs by inducing an antiviral response mediated by interferon that leads to general inhibition of protein synthesis and nonspecific degradation of mRNAs. Moreover, recent reports demonstrate that under certain conditions, short interfering RNAs (siRNAs, 21-25 bp) may activate the interferon system. Mouse oocytes and preimplantation embryos apparently lack this response, as potent and specific inhibition of gene expression triggered by long dsRNA is observed in these cells. In the present study, we analyzed the global pattern of gene expression by microarray analysis in transgenic mouse oocytes expressing long dsRNA and find no evidence of off-targeting. We also report that genes involved in the interferon response pathway are not expressed in mouse oocytes, even after exposure for an extended period of time to long dsRNA.

Animals↗
PubMed2005

RNA transcript profiling during zygotic gene activation in the preimplantation mouse embryo.

Zygotic gene activation is essential for development beyond the 2-cell stage in the preimplantation mouse embryo. Based on alpha-amanitin-sensitive BrUTP incorporation, transcription initiates in the 1-cell embryo and a major reprogramming of gene expression driven by newly expressed genes is prominently observed during the 2-cell stage. Superimposed on genome activation is the development of a transcriptionally repressive state that is mediated at the level of chromatin structure. The identity of the genes that are expressed during the 1- and 2-cell stages, however, is poorly described, as are those genes involved in mediating the transcriptionally repressive state. Using the Affymetrix MOE430 mouse GeneChip set, we characterized the set of alpha-amanitin-sensitive genes expressed during the 1- and 2-cell stages, and we used Expression Analysis Systematic Explorer (EASE) and Ingenuity Pathway Analysis (IPA) to identify biological and molecular processes represented by these genes, as well as interactions among them. We find that although the 1-cell embryo is transcriptionally active, we did not detect any transcripts present on the MOE430 GeneChip set to be alpha-amanitin-sensitive. Thus, what the BrUTP incorporation represents remains elusive. About 17% of genes expressed in the 2-cell embryo are alpha-amanitin-sensitive. EASE analysis reveals that genes involved in ribosome biogenesis and assembly, protein synthesis, RNA metabolism and transcription are over-represented, suggesting that genome activation during 2-cell stage may not be as global and promiscuous as previously proposed. IPA implicated Myc and Hdac1 as candidate genes involved in genome activation and the development of the transcriptionally repressive state, respectively.

Animals↗
PubMed2005

Egg activation events are regulated by the duration of a sustained [Ca2+]cyt signal in the mouse.

Although the dynamics of oscillations of cytosolic Ca2+ concentration ([Ca2+]cyt) play important roles in early mammalian development, the impact of the duration when [Ca2+]cyt is elevated is not known. To determine the sensitivity of fertilization-associated responses [i.e., cortical granule exocytosis, resumption of the cell cycle, Ca2+/calmodulin-dependent protein kinase II (CaMKII) activity, recruitment of maternal mRNAs] and developmental competence of the parthenotes to the duration of a [Ca2+]cyt transient, unfertilized mouse eggs were subjected to a prolonged [Ca2+]cyt change for 15, 25, or 50 min by means of repetitive Ca2+ electropermeabilization at 2-min intervals. The initiation and completion of fertilization-associated responses are correlated with the duration of time in which the [Ca2+]cyt is elevated, with the exception that autonomous CaMKII activity is down-regulated with prolonged elevated [Ca2+]cyt. Activated eggs from 25- or 50-min treatments readily develop to the blastocyst stage with no sign of apoptosis or necrosis and some implant. Ca2+ influx into unfertilized eggs causes neither Ca2+ release from intracellular stores nor rapid removal of cytosolic Ca2+. Thus, the total Ca2+ signal input appears to be an important regulatory parameter that ensures completion of fertilization-associated events and oocytes have a surprising degree of tolerance for a prolonged change in [Ca2+]cyt.

Animals↗
PubMed2005

Absence of the DNA-/RNA-binding protein MSY2 results in male and female infertility.

MSY2, a germ-cell-specific member of the Y-box family of DNA-/RNA-binding proteins, is proposed to function as a coactivator of transcription in the nucleus and to stabilize and store maternal and paternal mRNAs in the cytoplasm. In mice lacking Msy2, a normal Mendelian ratio is observed after matings between heterozygotes with equal numbers of phenotypically normal but sterile male and female homozygotes (Msy2-/-). Spermatogenesis is disrupted in postmeiotic null germ cells with many misshapen and multinucleated spermatids, and no spermatozoa are detected in the epididymis. Apoptosis is increased in the testes of homozygotes, and real-time RT-PCR assays reveal large reductions in the mRNA levels of postmeiotic male germ cell mRNAs and smaller reductions of meiotic germ cell transcripts. In females, there is no apparent decrease in either the number of follicles or their morphology in ovaries obtained from 2- and 8-day-old Msy2-/- mice. In contrast, follicle number and progression are reduced in 21-day-old Msy2-/- ovaries. In adult Msy2-/- females, oocyte loss increases, anovulation is observed, and multiple oocyte and follicle defects are seen. Thus, Msy2 represents one of a small number of germ-cell-specific genes whose deletion leads to the disruption of both spermatogenesis and oogenesis.

Animals↗
PubMed2005

Design of potent and selective 2-aminobenzimidazole-based p38alpha MAP kinase inhibitors with excellent in vivo efficacy.

We report the design and discovery of a 2-aminobenzimidazole-based series of potent and highly selective p38alphainhibitors. The lead compound 1 had low-nanomolar activity in both ATP competitive enzyme binding and inhibition of TNFalpha release in macrophages. Compound 18 showed excellent pharmacokinetics properties and oral activity in the rat collagen induced arthritis model compared with other p38 reference compounds. A SAR strategy to address CyP3A4 liability is also described.

Administration, Oral↗
PubMed2005

PAR-3 defines a central subdomain of the cortical actin cap in mouse eggs.

The evolutionarily conserved partitioning defective (PAR) protein PAR-3 is pivotal for establishing and maintaining cell polarity. During mammalian oocyte maturation, the radially symmetric oocyte is transformed into a highly polarized metaphase II (MII)-arrested egg. We therefore examined several aspects of PAR-3 expression during oocyte maturation. We cloned two novel PAR-3 transcripts from an oocyte library that likely encode proteins of Mr = 73 K and 133 K that are phosphorylated during maturation. PAR-3, which is found throughout the GV-intact oocyte, becomes asymmetrically localized during meiosis. Following germinal vesicle breakdown, PAR-3 surrounds the condensing chromosomes and associates with the meiotic spindles. Prior to emission of the first and second polar bodies, PAR-3 is located within a central subdomain of the polarized actin cap, which overlies the spindle. This cortical PAR-3 localization depends on intact microfilaments. These results suggest a role for PAR-3 in establishing asymmetry in the egg and in defining the future site of polar body emission.

Actin Cytoskeleton↗
PubMed2005

Phosphorylated MARCKS: a novel centrosome component that also defines a peripheral subdomain of the cortical actin cap in mouse eggs.

MARCKS (myristoylated alanine-rich C-kinase substrate) is a major substrate for protein kinase C (PKC), a kinase that has multiple functions during oocyte maturation and egg activation, for example, spindle function and cytoskeleton reorganization. We examined temporal and spatial changes in p-MARCKS localization during maturation of mouse oocytes and found that p-MARCKS is a novel centrosome component based its co-localization with pericentrin and gamma-tubulin within microtubule organizing centers (MTOCs). Like pericentrin, p-MARCKS staining at the MI spindle poles was asymmetric. Based on this asymmetry, we found that one end of the spindle was preferentially extruded with the first polar body. At MII, however, the spindle poles had symmetrical p-MARCKS staining. p-MARCKS also was enriched in the periphery of the actin cap overlying the MI or MII spindle to form a ring-shaped subdomain. Because phosphorylation of MARCKS modulates its actin crosslinking function, this localization suggests p-MARCKS functions as part of the contractile apparatus during polar body emission. Our finding that an activator of conventional and novel PKC isoforms did not increase the amount of p-MARCKS suggested that an atypical isoform was responsible for MARCKS phosphorylation. Consistent with this idea, immunostaining revealed that the staining patterns of p-MARCKS and the active form of the atypical PKC zeta/lambda isoform(s) were very similar. These results show that p-MARCKS is a novel centrosome component and also defines a previously unrecognized subdomain of the actin cap overlying the spindle.

Actins↗
PubMed2005

Role of MAP kinase and myosin light chain kinase in chromosome-induced development of mouse egg polarity.

During maturation, the mouse oocyte is transformed into a highly polarized egg, characterized by an actin cap and cortical granule-free domain (CGFD) overlying the meiotic spindle that is in close proximity to the cortex. The presence of spindle/chromosomes or microinjected sperm chromatin in the cortical region initiates this cortical reorganization, but the pathway is unknown. We report that cortical reorganization induced by microinjected sperm chromatin is blocked by inhibitors of microfilament assembly or disassembly. Active mitogen-activated protein kinase (MAPK), which becomes enriched in the region of sperm chromatin, is required for cortical reorganization, because microinjected sperm chromatin fails to induce cortical reorganization in Mos-/- eggs, which lack MAPK activity. Last, myosin light chain kinase (MLCK), which can be directly phosphorylated and activated by MAPK, appears involved, because the MLCK inhibitors ML-7 and Peptide 18 prevent sperm chromatin-induced cortical reorganization. These results provide new insights into how cortical reorganization occurs independently of extracellular signals to generate egg polarity.

Actins↗
PubMed2005

The DNA/RNA-binding protein MSY2 marks specific transcripts for cytoplasmic storage in mouse male germ cells.

During spermatogenesis, male germ cells temporally synthesize many proteins as they differentiate through meiosis and become spermatozoa. The germ cell Y-box protein, MSY2, constituting approximately 0.7% of total protein in male germ cells, binds to a consensus promoter element, and shows a general lack of RNA-binding specificity. Combining immunoprecipitation and suppressive subtractive hybridization, we identified populations of germ cell mRNAs that are not bound or bound by MSY2. The former population is enriched in cell growth and ubiquitously expressed mRNAs, whereas the latter population is enriched for stored or translationally delayed, male gamete-specific transcripts. Chromatin precipitation assays reveal that most of the MSY2 target mRNAs are transcribed from genes containing the Y-box DNA-binding motif in their promoters. In transgenic mice, mRNAs encoding exogenous GFP are directed or not directed into the MSY2-bound fraction by promoters containing or lacking the Y-box motif, respectively. We propose that MSY2 marks specific mRNAs in the nucleus for cytoplasmic storage, thereby linking transcription and mRNA storage/translational delay in meiotic and postmeiotic male germ cells of the mouse.

Animals↗