Menopause results from loss of ovarian function and marks the end of a woman's reproductive life. Alleles of the human SYCP2L locus are associated with age at natural menopause. SYCP2L is a paralogue of the synaptonemal complex... more
Menopause results from loss of ovarian function and marks the end of a woman's reproductive life. Alleles of the human SYCP2L locus are associated with age at natural menopause. SYCP2L is a paralogue of the synaptonemal complex protein SYCP2 and is expressed exclusively in oocytes. Here we report that SYCP2L localizes to centromeres of dictyate stage oocytes, which represent the limited pool of primordial oocytes that are formed perinatally and remain arrested till ovulation. Centromere localization of SYCP2L requires its C-terminal portion, which is missing in truncated variants resulting from low-frequency nonsense mutations identified in humans. Female mice lacking SYCP2L undergo a significantly higher progressive loss of oocytes with age compared to wild-type females and are less fertile. Specifically, the pool of primordial oocytes becomes more rapidly depleted in SYCP2L-deficient than in wild-type females, such that with aging, fewer oocytes undergo maturation in developin...
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RNA interference (RNAi) is a suitable method for sequence-specific post-transcriptional gene silencing in a number of model systems. The following protocol describes delivery of a double-stranded RNA (dsRNA) of choice into mouse one-cell... more
RNA interference (RNAi) is a suitable method for sequence-specific post-transcriptional gene silencing in a number of model systems. The following protocol describes delivery of a double-stranded RNA (dsRNA) of choice into mouse one-cell embryos by microinjection. Microinjected embryos can be cultured for up to 4 d, i.e., to the blastocyst stage. The efficiency of knockdown by RNAi can be assayed by quantitative reverse transcriptase (RT)-PCR (qPCR).
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During meiosis I (MI) in oocytes the maturation-associated decrease of histone acetylation is critical for normal meiotic progression and accurate chromosome segregation. RBBP4 is a component of several different histone deacetylase... more
During meiosis I (MI) in oocytes the maturation-associated decrease of histone acetylation is critical for normal meiotic progression and accurate chromosome segregation. RBBP4 is a component of several different histone deacetylase containing chromatin-remodeling complexes, but RBBP4's role in regulating MI is not known. Depleting RBBP4 in mouse oocytes resulted in multipolar spindles at metaphase I (Met I) with subsequent perturbed meiotic progression and increased incidence of abnormal spindles, chromosome misalignment, and aneuploidy at Met II. We attribute these defects to improper deacetylation of histones because histones H3K4, H4K8, H4K12, and H4K16 were hyperacetylated in RBBP4-depleted oocytes. Importantly, we show that RBBP4-mediated histone deacetylation is essential for regulating bipolar spindle assembly, at least partially, through promoting Aurora kinase C (AURKC) function. These results are the first to identify RBBP4 as a regulator of histone deacetylation duri...
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The discovery of RNA interference (RNAi) in 1998 ushered in a new era in biology. RNAi currently serves as a favorite approach for inhibition of gene function in many areas of research. This article provides a brief review of RNAi and... more
The discovery of RNA interference (RNAi) in 1998 ushered in a new era in biology. RNAi currently serves as a favorite approach for inhibition of gene function in many areas of research. This article provides a brief review of RNAi and discussion of the benefits and drawbacks of using long double-stranded RNA (dsRNA) in mammalian oocytes and early embryos. We also provide an introduction to protocols for RNAi experiments in mouse, including preparation and microinjection of dsRNA into mouse oocytes and early embryos, and preparation and testing of constructs for transgenic RNAi based on long hairpin RNA expression.
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The imprinted regulation of H19 and Insulin-like growth factor 2 expression involves binding of the vertebrate insulator protein, CCCTC binding factor (CTCF), to the maternally hypomethylated differentially methylated domain (DMD). How... more
The imprinted regulation of H19 and Insulin-like growth factor 2 expression involves binding of the vertebrate insulator protein, CCCTC binding factor (CTCF), to the maternally hypomethylated differentially methylated domain (DMD). How this hypomethylated state is maintained during oogenesis and the role of CTCF, if any, in this process are not understood. With the use of a transgenic RNA interference (RNAi)-based approach to generate oocytes with reduced amounts of CTCF protein, we found increased methylation of the H19 DMD and decreased developmental competence of CTCF-deficient oocytes. Our results suggest that CTCF protects the H19 DMD from de novo methylation during oocyte growth and is required for normal preimplantation development.
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Using reverse transcription-polymerase chain reaction (RT-PCR) with degenerate oligonucleotides corresponding to two highly conserved motifs within the protein kinase family of catalytic domains, we isolated a PCR fragment encoding a... more
Using reverse transcription-polymerase chain reaction (RT-PCR) with degenerate oligonucleotides corresponding to two highly conserved motifs within the protein kinase family of catalytic domains, we isolated a PCR fragment encoding a novel member of the testis-specific serine/threonine kinases (STK) from mouse male mixed germ cell mRNA. This PCR fragment recognized a 1020-bp transcript in male germ cells by northern blot analysis and was used to clone a full-length cDNA from a mouse mixed germ cell cDNA library. This cDNA has an open reading frame of 804 bases encoding a protein of 268 amino acids. This novel gene is almost identical to Stk22c, encoding a recently described testis-specific protein kinase, except for base-pair deletions that result in a shift in the coding region and an alteration of 22 amino acids (residues 109-131). Due to its homology with Stk22c, we have called this protein kinase gene Stk22d. Northern blot analysis revealed that this protein kinase is developmentally expressed in testicular germ cells and is not present in brain, ovary, kidney, liver, or early embryonic cells. We then cloned the human homologue of this protein kinase gene (STK22C) and found it to be expressed exclusively in the testis. Fluorescence in situ hybridization with both the human and mouse cDNA clones revealed syntenic localization on chromosomes 1p34-p35 and 4E1, respectively.
Research Interests: Information Systems, Genetics, Genomics, Gene expression, Humans, and 17 moreMice, Female, Animals, Male, tESTIS, Molecular cloning, Fluorescent in situ hybridization, Degeneration, Amino Acid Profile, Protein Kinase, Amino Acid Sequence, Base Sequence, Open Reading Frame, Spermatozoa, Germ Cell, Full Length Movies, and cDNA library
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The RNase III enzyme DICER generates both microRNAs (miRNAs) and endogenous short interfering RNAs (endo-siRNAs). Both small RNA species silence gene expression post-transcriptionally in association with the ARGONAUTE (AGO) family of... more
The RNase III enzyme DICER generates both microRNAs (miRNAs) and endogenous short interfering RNAs (endo-siRNAs). Both small RNA species silence gene expression post-transcriptionally in association with the ARGONAUTE (AGO) family of proteins. In mammals, there are four AGO proteins (AGO1-4), of which only AGO2 possesses endonucleolytic activity. siRNAs trigger endonucleolytic cleavage of target mRNAs, mediated by AGO2, whereas miRNAs cause translational repression and mRNA decay through association with any of the four AGO proteins. Dicer deletion in mouse oocytes leads to female infertility due to defects during meiosis I. Because mouse oocytes express both miRNAs and endo-siRNAs, this phenotype could be due to the absence of either class of small RNA, or both. However, we and others demonstrated that miRNA function is suppressed in mouse oocytes, which suggested that endo-siRNAs, not miRNAs, are essential for female meiosis. To determine if this was the case we generated mice tha...
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Specific mRNA degradation mediated by double-stranded RNA (dsRNA), which is termed RNA interference (RNAi), is a useful tool with which to study gene function in several systems. We report here that in mouse oocytes, RNAi provides a... more
Specific mRNA degradation mediated by double-stranded RNA (dsRNA), which is termed RNA interference (RNAi), is a useful tool with which to study gene function in several systems. We report here that in mouse oocytes, RNAi provides a suitable and robust approach to study the function of dormant maternal mRNAs. Mos (originally known as c-mos) and tissue plasminogen activator (tPA, Plat) mRNAs are dormant maternal mRNAs that are recruited during oocyte maturation; translation of Mos mRNA results in the activation of MAP kinase. dsRNA directed towards Mos or Plat mRNAs in mouse oocytes effectively results in the specific reduction of the targeted mRNA in both a time- and concentration-dependent manner. Moreover, dsRNA is more potent than either sense or antisense RNAs. Targeting the Mos mRNA results in inhibiting the appearance of MAP kinase activity and can result in parthenogenetic activation. Mos dsRNA, therefore, faithfully phenocopies the Mos null mutant. Targeting the Plat mRNA with Plat dsRNA results in inhibiting production of tPA activity. Finally, effective reduction of the Mos and Plat mRNA is observed with stoichiometric amounts of Mos and Plat dsRNA, respectively.
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Research Interests: DNA replication, Gene expression, Humans, Mice, Female, and 3 moreAnimals, Experimental, and Oocytes
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We generated mice lacking Cks2, one of two mammalian homologs of the yeast Cdk1-binding proteins, Suc1 and Cks1, and found them to be viable but sterile in both sexes. Sterility is due to failure of both male and female germ cells to... more
We generated mice lacking Cks2, one of two mammalian homologs of the yeast Cdk1-binding proteins, Suc1 and Cks1, and found them to be viable but sterile in both sexes. Sterility is due to failure of both male and female germ cells to progress past the first meiotic metaphase. The chromosomal events up through the end of prophase I are normal in both CKS2-/- males and females, suggesting that the phenotype is due directly to failure to enter anaphase and not a consequence of a checkpoint-mediated metaphase I arrest.
Research Interests: Science, Apoptosis, Multidisciplinary, In Situ Hybridization, Meiosis, and 17 moreMutation, Spermatogenesis, Mice, Female, Animals, Male, Sterilization, Male Infertility, tESTIS, Gene Targeting, Chromosome segregation, Genetic Recombination, Ovary, Epididymis, Oocytes, Female Infertility, and Germ Cell
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Fertilization triggers a rise in intracellular Ca(2+) concentration ([Ca(2+)](i)) in the egg that initiates a series of events known as egg activation. These events include cortical granule exocytosis that establishes a block to... more
Fertilization triggers a rise in intracellular Ca(2+) concentration ([Ca(2+)](i)) in the egg that initiates a series of events known as egg activation. These events include cortical granule exocytosis that establishes a block to polyspermy, resumption of meiosis, and recruitment of maternal mRNAs into polysomes for translation. Several calcium-dependent proteins, including calcium/calmodulin-dependent protein kinase II (CaMKII), have been implicated in egg activation. However, the precise role of CaMKII in mediating specific events of egg activation and the identity of the isoform(s) present in mouse eggs have not been unequivocally established. Through targeted deletion of the gamma isoform of CaMKII, we find that CaMKIIgamma is the predominant CaMKII isoform in mouse eggs and that it is essential for egg activation. Although CaMKIIgamma(-/-) eggs exhibit a normal pattern of Ca(2+) oscillations after insemination and undergo cortical granule exocytosis, they fail to resume meiosis or to recruit maternal mRNAs. Surprisingly, we find that the recruitment of maternal mRNAs does not directly depend on CaMKII, but requires elevated [Ca(2+)](i) and metaphase II exit. We conclude that CaMKIIgamma specifically controls mouse egg activation by regulating cell cycle resumption.
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In the preimplantation mouse embryo, activation of the embryonic genome is accompanied by a transient enrichment of histone H4 acetylated at lysines 5, 8, and 12 at the nuclear periphery (Worrad et al., 1995: Development 121:2949-2959).... more
In the preimplantation mouse embryo, activation of the embryonic genome is accompanied by a transient enrichment of histone H4 acetylated at lysines 5, 8, and 12 at the nuclear periphery (Worrad et al., 1995: Development 121:2949-2959). In the present report, we use laser-scanning confocal microscopy and a new panel of antibodies to define the distribution of specific acetylated isoforms of the other three core histones in mouse embryos at the 1- to 4-cell stage. We find that histone H3 acetylated at lysine 9 and/or 18 (H3.Ac9/18) and the single acetylated form of H2A (H2A.Ac5) become transiently enriched at the nuclear periphery in the 2-cell embryo. In contrast, H3.Ac14, H3.Ac23, and acetylated H2B, like H4.Ac16, remain distributed throughout the nucleoplasm. The staining intensity with antisera to H3.Ac9/18, even at the periphery was weak compared to that obtained with antisera to acetylated H4. A brief period of culture, however, in the presence of the inhibitor of histone deacetylases trichostatin A (TSA) or trapoxin increased labeling. Thus, the steady-state level of H3.Ac9/18 at the nuclear periphery and H3.Ac14 and H3.Ac23 in the nuclear interior is relatively low, but turnover remains high. The localization of selected acetylated isoforms of H3 and H2A at the nuclear periphery was independent of ongoing transcription or of cytokinesis, but did require DNA replication. We propose a model in which the selective, replication-dependent acetylation and deacetylation of zygotic chromatin at the nuclear periphery mediates the programming of zygotic transcription.
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Using reverse transcription-polymerase chain reaction (RT-PCR) with degenerate oligonucleotides corresponding to two highly conserved motifs within the protein kinase family of catalytic domains, we isolated a PCR fragment encoding a... more
Using reverse transcription-polymerase chain reaction (RT-PCR) with degenerate oligonucleotides corresponding to two highly conserved motifs within the protein kinase family of catalytic domains, we isolated a PCR fragment encoding a novel member of the testis-specific serine/threonine kinases (STK) from mouse male mixed germ cell mRNA. This PCR fragment recognized a 1020-bp transcript in male germ cells by northern blot analysis and was used to clone a full-length cDNA from a mouse mixed germ cell cDNA library. This cDNA has an open reading frame of 804 bases encoding a protein of 268 amino acids. This novel gene is almost identical to Stk22c, encoding a recently described testis-specific protein kinase, except for base-pair deletions that result in a shift in the coding region and an alteration of 22 amino acids (residues 109-131). Due to its homology with Stk22c, we have called this protein kinase gene Stk22d. Northern blot analysis revealed that this protein kinase is developmentally expressed in testicular germ cells and is not present in brain, ovary, kidney, liver, or early embryonic cells. We then cloned the human homologue of this protein kinase gene (STK22C) and found it to be expressed exclusively in the testis. Fluorescence in situ hybridization with both the human and mouse cDNA clones revealed syntenic localization on chromosomes 1p34-p35 and 4E1, respectively.
Research Interests: Information Systems, Genetics, Genomics, Gene expression, Humans, and 17 moreMice, Female, Animals, Male, tESTIS, Molecular cloning, Fluorescent in situ hybridization, Degeneration, Amino Acid Profile, Protein Kinase, Amino Acid Sequence, Base Sequence, Open Reading Frame, Spermatozoa, Germ Cell, Full Length Movies, and cDNA library
Research Interests: Gene regulation, Gene expression, Oogenesis, Biological Sciences, RNA interference, and 16 morePregnancy, Meiosis, Mice, Female, Animals, Male, Early development, Post-Transcriptional Gene Regulation, Regulatory Network, Oocyte Maturation, microRNAs, Genome Organization, Embryonic Development, Gene Expression Programming, Base Sequence, and Oocytes
The blastomere biopsy procedure does not affect preimplantation embryo development or global patterns of gene expression in a mouse model of Preimplantation Genetic Testing (PGT). However, zona breaching, which is inherent to the... more
The blastomere biopsy procedure does not affect preimplantation embryo development or global patterns of gene expression in a mouse model of Preimplantation Genetic Testing (PGT). However, zona breaching, which is inherent to the blastomere biopsy procedure, causes significant premature and sometimes abnormal hatching.
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Research Interests: Developmental Biology, Stem Cells, Cell Adhesion, DNA replication, Biological Sciences, and 15 moreCell Differentiation, Pregnancy, Basement Membrane, Mice, Female, Animals, Alternative splicing, Blastocyst, Developmental, Embryonic Development, Time Factors, Retinoic Acid, Alternative Splicing, Protein isoforms, and Embryos
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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... more
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.
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Research Interests: Developmental Biology, Biological Chemistry, Signal Transduction, Biological Sciences, Mice, and 25 moreCholesterol, cyclic AMP, Animals, Male, Biological, Signal Transduction Pathway Models, Phosphorylation, Developmental, CHEMICAL SCIENCES, Cattle, Fertilization, Time Dependent, Time Factors, High Density Lipoprotein, Cyclodextrins, Acrosome, Sterols, Spermatozoa, Bovine Serum Albumin, Protein Binding, Plasma Membrane, Tyrosine, Tyrosine Phosphorylation, Serum albumin, and Freeze-fracturing
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RNA interference (RNAi), the targeted mRNA degradation by double-stranded RNA (dsRNA), is a useful tool for studying gene function in several organisms. Here we report results of experiments with mammalian dsRNA expression vectors that... more
RNA interference (RNAi), the targeted mRNA degradation by double-stranded RNA (dsRNA), is a useful tool for studying gene function in several organisms. Here we report results of experiments with mammalian dsRNA expression vectors that are suitable to study gene function in mouse oocytes and preimplantation embryos. The plasmid vectors were constructed to contain the SV40 small intron, EGFP coding sequence to permit detection of expression, and an inverted repeat to mos mRNA that would form a hairpin dsRNA. Results of the experiments indicated that (i) hairpin dsRNA was just as effective as dsRNA (i.e., annealed sense and antisense RNA) in promoting the destruction of targeted mRNA, (ii) the EGFP marker could be expressed from the construct, and (iii) the distance of the SV40 intron from the inverted repeat was critical for the transcribed RNA to function in RNAi.
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The identity of mammalian genes involved in RNA interference (RNAi), the targeted sequence-specific mRNA degradation by double-stranded RNA (dsRNA), is poorly defined. Here we report the analysis of mice with null mutations of Wrn, Blm,... more
The identity of mammalian genes involved in RNA interference (RNAi), the targeted sequence-specific mRNA degradation by double-stranded RNA (dsRNA), is poorly defined. Here we report the analysis of mice with null mutations of Wrn, Blm, and RecQ1 genes that are related to Mut-7 and Qde3, two genes essential for RNAi in Caenorhabditis elegans and quelling in Neurospora, respectively. Our results suggest that Wrn, Blm, and RecQ1 are not involved in sequence-specific mRNA degradation in mammals in response to dsRNA, suggesting potential differences in the mammalian RNAi pathway.