Richard M. Schultz, Ph. D. Charles and William L. Day Distinguished Professor of Biology and SAS Associate Dean for the Natural Sciences Ph.D., Harvard University, 1975   204B Carolyn Lynch Laboratory Department of Biology University of Pennsylvania Philadelphia, PA 19104 USA V +1 215 898.7869 F +1 215 898.8780 E rschultz@sas.upenn.edu

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research : publications

Role of calcium oscillations during egg activation. It is well documented that changes in egg calcium homeostasis following sperm-egg fusion initiate events that constitute egg activation, e.g., cortical granule (CG) exocytosis, cell cycle resumption. In mammals, the release of intracellular calcium (Ca2+) following fertilization is mediated by inositol-1,4,5-trisphosphate (IP3) through the IP3 receptor. Fertilization-induced changes Ca2+ are characterized by an initial rise that lasts for several minutes, followed by a series of Ca2+ oscillations of shorter duration. The initial Ca2+ rise, as well as the subsequent oscillations, is due to the release of Ca2+ from an IP3-sensitive pool followed by Ca2+ sequestration. The ability of eggs to display this Ca2+ oscillatory behavior is acquired following oocyte maturation, and occurs only in the 1-cell embryo where it ceases following pronucleus formation. Although evidence suggests that these Ca2+ oscillations are critical for successful development, little is known about the underlying molecular mechanisms that link these oscillations to successful development. We are pursuing the linkage between these Ca2+ oscillations in the 1-cell embryo and recruitment and degradation of maternal mRNAs and changes in gene expression that occur during preimplantation development, and the linkage between these changes in gene expression and post-implantation development. This work is done in collaboration with Jean-Pierre Ozil. RNAi in mouse oocytes and preimplantation embryos Double-strand RNA (dsRNA) mediated post-transcriptional gene silencing, also known as RNA interference (RNAi), is a powerful tool to inhibit gene expression in several experimental model systems including Arabidopsis, C. elegans, and Drosophila. We have shown that RNAi operates in mouse oocytes and preimplantation embryos and have developed a transgenic RNAi approach that is suitable to study the function of any gene during mouse oocyte development and early embryogenesis. In addition we have been studying the role of Dicer in oocyte development and find that oocytes deficient in Dicer do not undergo proper maturation and that degradation of many maternal mRNAs does not occur correctly. We are currently determining the molecular basis for improper oocyte maturation in Dicer-deficient oocytes and deciphering a role for small RNAs non-coding RNAs in oocyte and preimplantation embryo development. This work is done in collaboration with Greg Hannon. Age-associated increase in incidence of aneuploidy A hallmark of animal development is an age-related decrease in fertility. In mammals, an increase in aneuploidy is the major underlying factor responsible for the increase in infertility with advancing age in human females. Aneuploidy is a leading cause of pregnancy loss, and when development goes to term, aneuploidy is an aggravating source of developmental disabilities and mental retardation, e.g., trisomy 21. The incidence of aneuploidy in eggs from women in their 20s’ is ~2%, but dramatically increases to 35% around 40 years-of-age. Spindle abnormalities and faulty chromosome congression on the metaphase plate are associated with advanced maternal age and likely contributes to the observed increased incidence of aneuploidy. Remarkably, little is known regarding the underlying molecular basis for the age-associated increase in aneuploidy. Mice also exhibit an increase in the incidence of aneuploidy with increasing maternal age. Expression profiling global patterns of gene expression of oocytes obtained from young and old mice revealed mis-expression of many genes, including genes involved the Spindle Assembly Checkpoint (SAC), chromosome congression and attachment to kinetocore microtubules, and spindle assembly. In addition, the normal pattern of degradation of maternal mRNAs was not observed following maturation of old oocytes. We are currently imaging individual oocytes obtained from young and old mice to determine the temporal and spatial dynamics of spindle assembly, spindle microtubule-kinetochore attachment, and chromosome congression and separation to determine whether old oocytes possess a weakened SAC. This work is done in collaboration with Michael Lampson. Regulation of degradation of maternal mRNA Degradation of maternal mRNA is thought to be essential to undergo the maternal-to-embryonic transition. Messenger RNA is extremely stable during oocyte growth in mouse and MSY2, an abundant germ cell-specific RNA-binding protein, likely serves as a mediator of global mRNA stability. Oocyte maturation, however, triggers an abrupt transition in which most mRNAs are significantly degraded. We noted that CDK1-mediated phosphorylation of MSY2 triggers this transition. Injecting Cdk1 mRNA, which activates CDK1, overcomes milrinone-mediated inhibition of oocyte maturation, induces MSY2 phosphorylation and the maturation-associated degradation of mRNAs. Inhibiting CDK1 following its activation with roscovitine inhibits MSY2 phosphorylation and prevents mRNA degradation. Expressing non-phosphorylatable dominant-negative forms of MSY2 inhibits the maturation-associated decrease in mRNAs, whereas expressing constitutively-active forms induces mRNA degradation in the absence of maturation and phosphorylation of endogenous MSY2. A positive-feedback loop of CDK1-mediated phosphorylation of MSY2 that leads to degradation of Msy2 mRNA that in turn leads to a decrease in MSY2 protein may ensure that the transition is irreversible. Current studies are addressing the developmental consequences of inhibiting degradation of maternal mRNAs that normally occurs during oocyte maturation. Effect of culture on gene expression and behavior The use of assisted reproductive technologies (ART) to treat human infertility is gaining widespread use, and it is estimated that in the US that 1 out of 80 children born in 2002 will have been conceived by ART. Disconcerting to many researchers is that the clinical procedures used in ART are rapidly outpacing the underlying science, an example of ART before science. We have noted that culture conditions can perturb global patterns of gene expression in preimplantation mouse embryos. In particular, certain culture conditions result in biallelic expression of the imprinted H19 gene in the blastocyst, and this biallelic expression persists in extra-embryonic tissue following implantation. Recent retrospective studies have unmasked an increased incidence of certain syndromes that are the result of loss-of-imprinting, highlighting the concern about the aggressive practice of ART. The long-term developmental and behavioral consequences of ART are unknown. To address this, we have developed a mouse model to study the effects of embryo culture, which is an integral part of every ART-conceived child, on behavior in the offspring. We find that mice derived from cultured embryos exhibit specific behavioral alterations in anxiety and spatial memory, e.g., mice derived from cultured embryos display deficiencies in spatial memory. We are pursuing these studies by (1) examining the effect of different culture conditions on the expression of a battery of imprinted genes, as well as on global patterns of gene expression as determined by microarray analysis, (2) altering the culture conditions to minimize or eliminate the behavioral consequences of culture, and (3) mimicking clinical procedures known to produce “low quality eggs” used in ART affect gene expression in the embryos and behavior in the offspring in our mouse model system. The work on imprinted gene expression is done collaboration with Marisa Bartolomei and the work on behavior is done in collaboration with Ted Abel.

selected publications Tseng, H., Chou, W., Zhang, X., Zhang, S., and Schultz, R.M. (2008). Mouse ribosomal RNA genes contain multiple differentially regulated variants. PLoS One. In Press. Solc, P., Saskova, A., Baran, V., Kubelka, M., Schultz, R.M., and Motlik, J. (2008). CDC25A phosphatase controls meiosis I progression in mouse oocytes. Dev. Biol. In Press. Tam, O.H., Aravin, A.A., Stein, P., Girard, A., Murchison, E.P., Cheloufi, S., Hodges, E., Anger, M., Sachidanandam, R., Schultz, R.M., and Hannon, G.J. (2008). Pseudogene-derived siRNAs regulate gene expression in mouse oocytes. Nature. In Press. Ma, P. and Schultz, R.M. (2008). Histone deacetylase 1 (HDAC1) regulates histone acetylation, development, and gene expression in preimplantation mouse embryos. Dev. Biol. In Press. Rivera, R.M., Stein, P., Weaver, J.R., Mager, J., Schultz, R.M., and Bartolomei, M.S. (2008). Manipulations of mouse embryos prior to implantation result in aberrant expression of imprinted genes on day 9.5 of development. Hum. Mol. Genet. 17, 1-14. Pan, H., Ma, P., Zhu, W., and Schultz, R.M. (2008). Age-associated increase in aneuploidy and changes in gene expression in mouse eggs. Dev. Biol. 316, 397-407. Murchison, E.P., Stein, P., Xuan, Z., Pan, H., Zhang, M.Q., Schultz, R.M., and Hannon, G.J. (2007). Critical roles for Dicer in the female germline. Genes and Development 21, 682-693. Vassena, R., Han, Z., Gao, S., Baldwin, D.A., Schultz, R.M., and Latham, K.E. (2007). Tough beginnings: alterations in the transcriptome of cloned embryos during the first two cell cycle. Dev. Biol. 304, 75-89. Reese, K.J., Lin, S., Verona, R.I., Schultz, R.M., and Bartolomei, M.S. (2007). Maintenance of paternal methylation and repression of the imprinted H19 gene requires MBD3. PLoS Genetics. PLoS Genetics 3, e137. Igarashi, H., Knott, J.G., Schultz, R.M., and Williams, C.J. (2007). Alterations in PLCb1 in mouse eggs change calcium oscillatory behavior following fertilization. Dev. Biol. 312, 321-330. Deng, M., Suraneini, P., Schultz, R.M., and Li, R. (2007). The Ran GTPase mediates chromatin signaling to control cortical polarity during polar body extrusion in mouse oocytes. Dev. Cell. 12, 301-308. Ozil, J.P., Banrezes, B., Szabolcs, T., Pan, H., and Schultz, R.M. (2006). Ca2+ oscillatory pattern in fertilized mouse eggs affects gene expression and development to term. Dev. Biol. 300, 534-544. Knott, J.T., Gardner, A.J., Madgwick, S., Jones, K.T., Williams, C.W., and Schultz, R.M. (2006). Calmodulin-dependent protein kinase II triggers mouse egg activation and embryo development in the absence of Ca2+ oscillations. Dev. Biol. 296, 388-395. Bultman, S.J., Gebur, T.C., Pan, H., Svoboda, P., Schultz, R.M., and Magnuson, T. (2006). Maternal BRG1 regualates zygotic genome activation in the mouse. Genes Dev. 20, 1744-1754. Ma, J., Zeng, F., Schultz, R.M., and Tseng, H. (2006). Basonuclin: A novel mammalian maternal-effect gene. Development 133, 2053-2062. Deng, M., Williams, C.J., and Schultz, R.M. (2005). Role of MAP kinase and myosin light chain kinase in chromosome-induced development of mouse egg polarity. Dev. Biol. 278, 358-366. Knott, J.G., Kurokawa, M., Fissore, R.A., Schultz, R.M., and Williams, C.J. (2005). Transgenic RNAi reveals role for mouse sperm phospholipase C-zeta in triggering Ca2+ oscillations during fertilization. Biol. Reprod. 72, 992-996. Michaut, M.A., Williams, C.J., and Schultz, R.M. (2005). Phosphorylated MARCKS: A novel centrosome component that also defines a peripheral subdomain of the cortical actin cap in mouse eggs. Dev. Biol. 280, 26-37. Duncan, F.E., Moss, S.B., Schultz, R.M., and Williams, C.J. (2005). PAR-3 defines a central subdomain of the cortical actin cap in mouse eggs. Dev. Biol. 280, 38-47. Ozil, J.-P., Markoulaki, S., Toth, S., Matson, S., Banrezes, B., Knott, J.G., Schultz, R.M., Huneau, D., and Ducibella, T. (2005). Egg activation events are regulated by the duration of a sustained [Ca2+]cyt signal in the mouse. Dev. Biol. 282, 39-54. Yan, W., Ma, L., Stein, S., Pangas, S.A., Burns, K.H., Bai, Y., Schultz, R.M., and Matzuk, M.M. (2005). An oocyte-specific oligoadenylate synthetase-like protein, OAS1D, inhibits the interferon/oligoadenylate synthetase/RNase L-mediated pathway in mouse oocytes. Mol. Cell Biol. 25, 4615-4624. Yang, J., Medvedev, S., Yu, J., Tang, L.C., Agno, J.E., Matzuk, M.M., Schultz, R.M., and Hecht, N.B. (2005). Absence of the DNA/RNA-binding protein MSY2 results in male and female infertility. Proc. Natl. Acad. Sci. USA 102, 5755-5760. Zeng, F., and Schultz, R.M. (2005). RNA transcript profiling during zygotic gene activation in the preimplantation mouse embryo. Dev. Biol. 283, 40-57. Pan, H., O’Brien, M.J., Wigglesworth, K., Eppig, J.J., and Schultz, R.M. (2005). Transcript profiling during mouse oocyte development and the effect of gonadotropin priming and development in vitro. Dev. Biol. 286, 493-506. Stein, P., Zeng, F., Pan, H., and Schultz, R.M. (2005). Absence of non-specific effects of RNA interference triggered by long double-stranded RNA in mouse oocytes. Dev. Biol. 286, 464-471. Fedoriw, A.M., Stein, P., Svoboda, P., Schultz, R.M., and Bartolomei, M.S. (2004). Maternal CTCF requirement for appropriate DNA methylation of the imprinted H19 gene. Science 303, 238-240. Ecker, D.J., Stein, P., Xu, Z., Williams, C.J., Kopf, G.S., Bilker, W.B., Abel, T., and Schultz, R.M. (2004). Long-term effects of culture of preimplantation mouse embryos on behavior. Proc. Natl. Acad. Sci. USA. 101, 1595-1600. Yu, J., Deng, M., Medvedev, S., Yang, J., Hecht, N.B., and Schultz, R.M. (2004). Transgenic RNAi-meidaed reduction of MSY2 in mouse oocytes results in reduced fertility. Dev. Biol. 268, 195-206. Svoboda, P., Stein, S., Anger, M., Bernstein, E., Hannon, G.J., and Schultz, R.M. (2004). RNAi and expression of retrotransposons MuERV-L and IAP in preimplantation mouse embryos. Dev. Biol. 269, 276-285. Mann, M.R.W., Lee, S.S., Doherty, A.S., Verona R.I., Nolen, L.D., Schultz, R.M. and Bartolomei, M.S. (2004). Selective loss of imprinting in the placenta following preimplantation development in culture. Development. 131, 3727-3735. Zeng, F., Baldwin, D.A., and Schultz, R.M. (2004). Transcript profiling during preimplantation mouse development. Dev. Biol., 272, 483-496. Svoboda, P., Stein, P., Filipowicz, W., and Schultz, R.M. (2004). Lack of homologous sequence-specific DNA methylation in response to stable dsRNA expression in mouse oocytes. Nucl. Acids Res. 32, 3601-3606. Xu, Z., Williams, C.J., Kopf, G.S., and Schultz, R.M. (2003). Maturation-associated increase in IP3 receptor type 1: Role in conferring increased IP3 sensitivity and Ca2+ oscillatory behavior in mouse eggs. Dev. Biol. 254, 163-171. Stein, P., Svoboda, P., Anger, M., and Schultz, R.M. (2003). RNAi: Mammalian oocytes do it without RNA-dependent RNA polymerase. RNA 9, 187-192. Yu, J., Hecht, N.B., and Schultz, R.M. (2003). Requirement for RNA-binding activity of MSY2 for cytoplasmic localization and retention in mouse oocytes. Dev. Biol. 255, 249-262. Ducibella, T., Huneau, D., Angelichio, E., Xu, Z., Schultz, R.M., Kopf, G.S., Fissore, R., Madoux, S., and Ozil, J.-P. (2002). Egg to embryo transition is driven by differential responses to Ca2+ oscillation number. Dev. Biol. 250, 280-291. 189 Stein, P., Svoboda, S., and Schultz, R.M. (2003). Transgenic RNAi in mouse oocytes: A simple and fast approach to study gene function. Dev. Biol. 256, 187-193.