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

Publication-qual. TIFF (~0.5 MB)

research : publications

Molecular mechanisms of egg activation. Changes in egg calcium homeostasis following sperm-egg fusion initiate events that constitute egg activation, e.g., cortical granule (CG) exocytosis, cell cycle resumption. Fertilization-induced changes Ca2+ are characterized by an initial rise in Ca2+ 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, occurs only in the 1-cell embryo, and ceases following pronucleus formation. Although 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 how Ca2+ oscillations are linked to recruitment/degradation of maternal mRNAs, as well as changes in gene expression that occur during pre- and post-implantation development. In addition, we are exploring the role of CaMKII-gamma in integrating the calcium signal. Current studies focus on whether the sole function of CaMKII-gamma is to mediate exit from the arrest at metaphase II. 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 studies centered on the role of Dicer in oocyte development have revealed that oocytes deficient in Dicer do not undergo proper maturation and that degradation of many maternal mRNAs does not occur correctly. Recently we noted that in contrast to somatic cells, endogenous miRNAs in fully-grown GV oocytes poorly repressed translation of mRNA reporters whereas their RNAi-like activity was much less affected. In addition, reporter mRNA carrying let-7-binding sites failed to localize to P-body-like structures in oocytes. These findings indicate that miRNA-mediated degradation of maternal mRNAs is not a robust pathway in oocytes and that the failure of Dicer-deficient oocytes to mature properly can be ascribed to failure to generate endo-siRNAs. Current studies are focused on deciphering the molecular mechanisms that why this pathway does not function robustly in oocytes and what pathways are involved in degradation of maternal mRNAs that initiates with the onset of oocyte maturation (see below). Maternal 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. Results of these studies have demonstrated that old oocytes possess a highly functional SAC, i.e., a defective SAC is unlikely the primary cause of aneuploidy associated with maternal age. Noteworthy, is our recent finding that ~90% of age-related aneuploidies are best explained by weakened centromere cohesion. Moreover, levels of the meiotic cohesin protein Rec8 are severely reduced on chromosomes in oocytes from old mice. Together, these results demonstrate that the maternal age-associated increase in aneuploidy is primarily due to a failure to effectively replace cohesin proteins that are lost from chromosomes during aging. 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 Murai, S., Stein, P., Buffone, M.B., Yamashita, S., and Schultz, R.M. (2010) Recuritment of Orc6I, a dormant maternal mRNA in mouse oocytes, is essential for DNA replication in 1-cell embryos. Dev. Biol. In Press. Flemr, M., Ma, J., Schultz, R.M., and Svoboda, P. (2009). P-body loss is concomitant with formation of an mRNA storage domain in mouse oocytes. Biol. Reprod. In Press. Ma, Jun, Flemr, M., Stein, P., Berninger, P., Malik, R., Zavolan, M., Svoboda, P., and Schultz, R.M. (2009). microRNA activity is suppressed in mouse oocytes. Curr. Biol. 9, 265-270. Backs, J., Stein, P., Backs, T., Duncan, F.E., Grueter, C.E., McAnnally, J., Qi, X., Schultz, R.M. and Olson, E.N. (2009). The γ isoform of CaM kinase II controls mouse egg activation by regulating cell cycle resumption. Proc. Natl. Acad. Sci. USA. 107, 81-86. Buffone, M.G., Schindler, K., and Schultz, R.M. (2009). Over-expression of CDC14B causes mitotic arrest and inhibits zygotic genome activation in mouse preimplantation embryos. Cell Cycle. 8, 3904-3913. Duncan, F.E., Chiang, T., Schultz, R.M., and Lampson, M.A. (2009). Evidence that a defective spindle assembly checkpoint is not the primary cause of maternal age-associated aneuploidy in mouse eggs. Biol. Reprod. 81, 768-776. Shuda, K., Schindler, K., Ma, J., Schultz, R.M., and Donovan, P.J. (2009). Aurora kinase B modulates chromosome alignment in mouse oocytes. Mol. Reprod. Dev. 76, 1094-1105. Schindler, K. and Schultz, R.M. (2009). The CDC14A phosphatase regulates oocyte maturation in mouse. Cell Cycle. 8, 1090-1098. Schindler, K. and Schultz, R.M. (2008). CDC14B acts through FZR1 (CDH1) to prevent meiotic maturation of mouse oocytes. Biol. Reprod. 80, 795-803. Ihara, M., Stein, P., and Schultz, R.M. (2008). UBC9, a SUMO-conjugating enzyme, localizes to nuclear speckles and stimulates transcription in mouse oocytes. Biol. Reprod. 79, 2368-2376. Wan, L-B., Pan, H., Hannenhalli, S., Cheng, Y., Ma, J., Fedoriw, A., Lobanenkov, V., Latham, K.E., Schultz, R.M., and Bartolomei, M.S. (2008). Maternal depletion of CTCF reveals multiple functions during oocyte and preimplantation embryo development. Development 135, 2729-2738 Medvedev, S., Yang, J., Hecht, N.B., and Schultz, R.M. (2008). CDC2A (CDK1)-mediated phosphorylation of MSY2 triggers maternal mRNA degradation during mouse oocyte maturation. Dev. Biol. 321, 205-215. Duncan, F.E., Stein, P., Williams, C.J., and Schultz, R.M. (2008). The effect of blastomere biopsy on preimplanation mouse embryo development and global gene expression. Fertil. Stert. 91, 1462-1465. Saskova, A., Solc, P., Vladimir, B., Kubelka, M., Schultz, R.M., and Motlik, J. (2008). Aurora Kinase A controls meiosis I progression in mouse oocytes. Cell Cycle, 7, 2368-2376. Ma, P. and Schultz, R.M. (2008). Histone deacetylase 1 (HDAC1) regulates histone acetylation, development, and gene expression in preimplantation mouse embryos. Dev. Biol. 319, 110-120. 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. 453, 534-538. 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. 317, 260-269. Tseng, H., Chou, W., Zhang, X., Zhang, S., and Schultz, R.M. (2008). Mouse ribosomal RNA genes contain multiple differentially regulated variants. PLoS One. E1843. Pan, H., Ma, P., Zhu, W., and Schultz, R.M. (2008). Age-associated increase in aneuploidy and changes in gene expression in moue eggs. Dev. Biol. 316, 397-407. 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. 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. 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. 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. 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. Yang, J., Morales, C.R., Medvedev, S., Schultz, R. M., and Hecht, N.B. (2007). In the absence of the mouse DNA/RNA-binding protein MSY2 messenger RNA instability leads to spermatogenic arrest. Biol. Reprod. 76, 48-54. 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.