Heat shock protein expression during gametogenesis and embryogenesis.
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When cells are subjected to various stress factors, they increase the production of a group of proteins called heat shock proteins (hsp). Heat shock proteins are highly conserved proteins present in organisms ranging from bacteria to man. Heat shock proteins enable cells to survive adverse environmental conditions by preventing protein denaturation. Thus the physiological and pathological potential of hsps is enormous and has been studied widely over the past two decades. The presence or absence of hsps influences almost every aspect of reproduction. They are among the first proteins produced during mammalian embryo development. In this report, the production of hsps in gametogenesis and early embryo development is described. It has been suggested that prolonged and asymptomatic infections trigger immunity to microbial hsp epitopes that are also expressed in man. This may be relevant for human reproduction, since many couples with fertility problems have had a previous genital tract infection. Antibodies to bacterial and human hsps are present at high titers in sera of many patients undergoing in vitro fertilization. In a mouse embryo culture model, these antibodies impaired the mouse embryo development at unique developmental stages. The gross morphology of these embryos resembled cells undergoing apoptosis. The TUNEL (terminal deoxynucleotidyl transferase-mediated X-dUTP nick end labeling) staining pattern, which is a common marker of apoptosis, revealed that embryos cultured in the presence of hsp antibodies stained TUNEL-positive more often than unexposed embryos. These data extend preexisting findings showing the detrimental effect of immune sensitization to hsps on embryo development. (+info)
Increased instability of human CTG repeat tracts on yeast artificial chromosomes during gametogenesis.
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Expansion of trinucleotide repeat tracts has been shown to be associated with numerous human diseases. The mechanism and timing of the expansion events are poorly understood, however. We show that CTG repeats, associated with the human DMPK gene and implanted in two homologous yeast artificial chromosomes (YACs), are very unstable. The instability is 6 to 10 times more pronounced in meiosis than during mitotic division. The influence of meiosis on instability is 4.4 times greater when the second YAC with a repeat tract is not present. Most of the changes we observed in trinucleotide repeat tracts are large contractions of 21 to 50 repeats. The orientation of the insert with the repeats has no effect on the frequency and distribution of the contractions. In our experiments, expansions were found almost exclusively during gametogenesis. Genetic analysis of segregating markers among meiotic progeny excluded unequal crossover as the mechanism for instability. These unique patterns have novel implications for possible mechanisms of repeat instability. (+info)
The role of apoptosis in normal and abnormal embryonic development.
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Programmed cell death or apoptosis is a widespread biological phenomenon. Apoptosis is characterized by typical cell features such as membrane blebbing, chromatin condensation, and DNA fragmentation. It involves a number of membrane receptors (e.g., Fas, TNFR) and a cascade of signal transduction steps resulting in the activation of a number of cysteine proteases known as caspases. Disordered apoptosis may lead to carcinogenesis and participates in the pathogenesis of Alzheimer disease, Parkinson disease, or AIDS. Programmed cell death plays an important role in the processes of gamete maturation as well as in embryo development, contributing to the appropriate formation of various organs and structures. Apoptosis is one of the mechanisms of action of various cytotoxic agents and teratogens. Teratogen-induced excessive death of embryonic cells is undoubtedly one of the most important events preceding the occurrence of structural abnormalities, regardless of their nature. Therefore understanding the mechanisms involved in physiological as well as in disturbed or dysregulated apoptosis may lead to the development of new methods of preventive treatment of various developmental abnormalities. The present review summarizes data on the mechanisms of programmed cell death and concentrates on apoptosis involved in normal or disturbed gametogenesis and in normal and abnormal embryonic development. (+info)
EGO-1 is related to RNA-directed RNA polymerase and functions in germ-line development and RNA interference in C. elegans.
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BACKGROUND: Cell-fate determination requires that cells choose between alternative developmental pathways. For example, germ cells in the nematode worm Caenorhabditis elegans choose between mitotic and meiotic division, and between oogenesis and spermatogenesis. Germ-line mitosis depends on a somatic signal that is mediated by a Notch-type signaling pathway. The ego-1 gene was originally identified on the basis of genetic interactions with the receptor in this pathway and was also shown to be required for oogenesis. Here, we provide more insight into the role of ego-1 in germ-line development. RESULTS: We have determined the ego-1 gene structure and the molecular basis of ego-1 alleles. Putative ego-1 null mutants had multiple, previously unreported defects in germ-line development. The ego-1 transcript was found predominantly in the germ line. The predicted EGO-1 protein was found to be related to the tomato RNA-directed RNA polymerase (RdRP) and to Neurospora crassa QDE-1, two proteins implicated in post-transcriptional gene silencing (PTGS). For a number of germ-line-expressed genes, ego-1 mutants were resistant to a form of PTGS called RNA interference. CONCLUSIONS: The ego-1 gene is the first example of a gene encoding an RdRP-related protein with an essential developmental function. The ego-1 gene is also required for a robust response to RNA interference by certain genes. Hence, a protein required for germ-line development in C. elegans may be a component of the RNA interference/PTGS machinery. (+info)
Sexual stage-specific expression of a third calcium-dependent protein kinase from Plasmodium falciparum.
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A third calcium-dependent protein kinase (CDPK) gene has been isolated from the human malaria parasite Plasmodium falciparum by vectorette technology. The gene consists of five exons and four introns. The open reading frame resulting from removal of the four introns encodes a protein of 562 amino acid residues with a predicted molecular mass of 65.3 kDa. The encoded protein, termed PfCDPK3, consists of four distinct domains characteristic of a member of the CDPK family and displays the highest homology (46% identity and 69% similarity) to PfCDPK2, the second CDPK of P. falciparum. The N-terminal variable domain is rich in serine/threonine and lysine and contains multiple consensus phosphorylation sites for a range of protein kinases. The catalytic domain possesses all conserved motifs of the protein kinase family except for the highly conserved glutamic acid residue in subdomain VIII, which is replaced by a glutamine residue. The sequence of the junction domain comprising 31 amino acid residues is less conserved. The calmodulin-like regulatory domain contains four EF-hand calcium-binding motifs, each consisting of a loop of 12 amino acid residues which is flanked by two alpha-helices. Southern blotting of genomic DNA digests showed that the Pfcdpk3 gene is present as a single copy per haploid genome. A 2900 nucleotide transcript of this gene is expressed specifically in the sexual erythrocytic stage, indicating that PfCDPK3 is involved in sexual stage-specific events. It is proposed that PfCDPK3 may serve as a link between calcium and gametogenesis of P. falciparum. (+info)
Scorpine, an anti-malaria and anti-bacterial agent purified from scorpion venom.
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A novel peptide, scorpine, was isolated from the venom of the scorpion Pandinus imperator, with anti-bacterial activity and a potent inhibitory effect on the ookinete (ED(50) 0.7 microM) and gamete (ED(50) 10 microM) stages of Plasmodium berghei development. It has 75 amino acids, three disulfide bridges with a molecular mass of 8350 Da. Scorpine has a unique amino acid sequence, similar only to some cecropins in its N-terminal segment and to some defensins in its C-terminal region. Its gene was cloned from a cDNA library. (+info)
Regulated targeting of a protein kinase into an intact flagellum. An aurora/Ipl1p-like protein kinase translocates from the cell body into the flagella during gamete activation in chlamydomonas.
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In the green alga Chlamydomonas reinhardtii flagellar adhesion between gametes of opposite mating types leads to rapid cellular changes, events collectively termed gamete activation, that prepare the gametes for cell-cell fusion. As is true for gametes of most organisms, the cellular and molecular mechanisms that underlie gamete activation are poorly understood. Here we report on the regulated movement of a newly identified protein kinase, Chlamydomonas aurora/Ipl1p-like protein kinase (CALK), from the cell body to the flagella during gamete activation. CALK encodes a protein of 769 amino acids and is the newest member of the aurora/Ipl1p protein kinase family. Immunoblotting with an anti-CALK antibody showed that CALK was present as a 78/80-kDa doublet in vegetative cells and unactivated gametes of both mating types and was localized primarily in cell bodies. In cells undergoing fertilization, the 78-kDa CALK was rapidly targeted to the flagella, and within 5 min after mixing gametes of opposite mating types, the level of CALK in the flagella began to approach levels normally found in the cell body. Protein synthesis was not required for targeting, indicating that the translocated CALK and the cellular molecules required for its movement are present in unactivated gametes. CALK was also translocated to the flagella during flagellar adhesion of nonfusing mutant gametes, demonstrating that cell fusion was not required for movement. Finally, the requirement for flagellar adhesion could be bypassed; incubation of cells of a single mating type in dibutyryl cAMP led to CALK translocation to flagella in gametes but not vegetative cells. These experiments document a new event in gamete activation in Chlamydomonas and reveal the existence of a mechanism for regulated translocation of molecules into an intact flagellum. (+info)
H19 and Igf2 monoallelic expression is regulated in two distinct ways by a shared cis acting regulatory region upstream of H19.
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H19 and Igf2 are expressed in a monoallelic fashion from the maternal and paternal chromosomes, respectively. A region upstream of H19 has been shown to regulate such imprinted expression of both genes in cis. We have taken advantage of a loxP/cre recombinase-based strategy to delete this region in mice in a conditional manner to determine the temporal requirement of the upstream region in initiating and maintaining the imprinted expression of H19 and Igf2. Analysis of allele-specific expression of H19 and Igf2 and DNA methylation at the H19 promoter demonstrates that this region controls the monoallelic expression of the two genes in different ways, suggesting that it harbors two functionally distinct regulatory elements. Continued presence of the region is required to silence maternal Igf2 in accordance with its proposed role as an insulator. However, it does not have a direct role in keeping the paternal H19 promoter silenced. Instead, on the paternal chromosome, the upstream element mediates epigenetic modifications of the H19 promoter region during development, leading to transcriptional silencing of H19. Thereafter, its presence is redundant for preventing transcription. Presently, this temporal requirement of the silencing element appears to be a unique cis activity in the mammalian system. However, it is likely that other cis-acting elements, positive and negative, have the ability to effect stable changes in the chromatin structure and are not constantly required to give signals to the transcriptional machinery. (+info)