Mechanism of mammalian ovulation

The sequence of events within the ovary during the process of ovulation discussed in this review is schematically represented in Fig. 1. It is obvious that LH, perhaps with some contribution from FSH, is the normal physiological trigger for the ovulatory sequence of events, and it appears from the available information that the effects of LH are mainly mediated via adenylate cyclase and increased cAMP levels. The cAMP in turn, via cAMP-dependent protein kinase, influences at least three distinct steps in the ovulatory process which seem to be of crucial importance, namely 1) the stimulation of steroidogenesis; 2) the stimulation of cyclooxygenase/lipooxygenase leading to increased prostaglandin/leukotriene synthesis; and 3) the stimulation of plasminogen activator which catalyzes the conversion of plasminogen to plasmin. A fourth crucial step in the ovulatory mechanism is the LH-induced increase in latent collagenase, but it remains to be determined if this step is mediated via cAMP. Concomitant with the increase in latent collagenase, there also appears to be an LH-dependent increase in collagenase inhibitors. The latent collagenase is then activated, and it appears that leukotrienes and prostaglandins, as well as plasmin, may be involved in this process. The active collagenase causes a digestion of the collagen in the follicle wall, and plasmin, as well as possibly other proteolytic enzymes such as proteoglycanases, may cause a further dissociation of the follicular wall. These processes of digestion of collagen and dissociation of the collagen fibers result in an opening in the follicular wall with the formation of the stigma and rupture. While the weakening of the follicular wall takes place throughout the entire wall, rupture remains for the most part a localized process at the apex of the follicle. This localization of the rupture may be explained on the basis of mechanical factors operating when the follicle wall thins and weakens. While it is clear that prostaglandins and leukotrienes can influence smooth muscle by causing contractions and that these compounds can cause vascular changes such as increased permeability, vasodilation, and vasoconstriction, it is not clear what the exact role of these latter processes are in ovulation. It appears that progesterone and not estrogen play an important role in the mechanism of LH-induced follicular rupture, but the locus of action of progesterone and its mechanism of action remains to be determined.(ABSTRACT TRUNCATED AT 400 WORDS)     

Biology of mammalian Isospora

Isospora species are coccidial parasites that can cause serious disease in humans and pigs. Disease is observed less frequently in non-human primates, dogs or cats. Isospora species do not produce disease in horses, domestic ruminants or domestic poultry, and reports of isosporan oocysts in the feces of these hosts probably represents pseudoparasites that originated in feed or water contaminated with wild bird feces. David Lindsay and Byron Blagburn here summarize what is known about the biology of the Isospora species of domestic animals and non-human primates.     

Immunochemistry of mammalian cholinesterases

Advances in the study of cholinesterase biology have been facilitated by the development of polyclonal and monoclonal antibodies to acetylcholinesterase (AChE) (EC 3.1.1.7) and butyrylcholinesterase (BuChE) (EC 3.1.1.8) in several laboratories. Our work has focused on murine monoclonal antibodies to the mammalian enzymes. Two dozen antibodies are now in hand, with primary specificity for the AChE of human red blood cells, rabbit brain, and rat brain, and for the BuChE of human plasma. These antibodies exhibit a restricted but useful range of affinities for other mammalian cholinesterases of corresponding types. Several applications are described, including an analysis of BuChE phylogeny within the higher primates, an immunodisplacement assay of AChE in normal human red blood cells and cells from patients with paroxysmal nocturnal hemoglobinuria, a study of immunochemical differences between membrane-associated and soluble AChE of rabbit brain, and initial work on the immunofluorescence cytochemistry of the rat brain.     

Organization of mammalian cytoplasm

Although the role of macromolecular interactions in cell function has attracted considerable attention, important questions about the organization of cells remain. To help clarify this situation, we used a simple protocol that measures macromolecule release after gentle permeabilization for the examination of the status of endogenous macromolecules. Treatment of Chinese hamster ovary cells with saponin under carefully controlled conditions allowed entry of molecules of at least 800 kDa; however, there were minimal effects on internal cellular architecture and protein synthesis remained at levels comparable to those seen with intact cells. Most importantly, total cellular protein and RNA were released from these cells extremely slowly. The release of actin-binding proteins and a variety of individual cytoplasmic proteins mirrored that of total protein, while marker proteins from subcellular compartments were not released. In contrast, glycolytic enzymes leaked rapidly, indicating that cells contain at least two distinct populations of cytoplasmic proteins. Addition of microfilament-disrupting agents led to rapid and extensive release of cytoplasmic macromolecules and a dramatic reduction in protein synthesis. These observations support the conclusion that mammalian cells behave as highly organized, macromolecular assemblies (dependent on the actin cytoskeleton) in which endogenous macromolecules normally are not free to diffuse over large distances.     

Centromeres of mammalian chromosomes

The centromere is the major cis-acting genetic locus involved in chromosome segregation in mitosis and meiosis. The mammalian centromere is characterized by large amounts of tandemly repeated satellite DNA and by a number of specific centromere proteins, at least one of which has been shown to interact directly with centromeric satellite DNA sequences. Although direct functional assays of chromosome segregation are still lacking, the data are most consistent with a structural and possibly functional role for satellite DNA in the mammalian centromere.     

Cryopreservation of mammalian embryos

As an innovative method for embryo cryopreservation, vitrification not only reduced the cooling stage duration to a minimum, but also eliminated any injuries cased by extracellular ice, which is a major cause of cell injury. Therefore, if embryos are treated adequately, high survival can be obtained. As a component of a vitrification solution, a permeating cryoprotective agent is essential, and additional inclusion of a macromolecule and a small saccharide makes the solution more effective. The author’s group composed a solution, designated EFS40, with ethylene glycol. Ficoll, and sucrose. This solution proved effective for the cryopreservation of various stages of embryos in many species. In this article, the author describes the detailed procedure for the vitrification of mouse morulae; related information is also described.     

DNA-fluorometry of mammalian sperm

Summary The DNA-content of sperm and testicular cells was measured by pulse-cytophotometry with high resolution. From flat sperm symmetric and narrow fluorescence distributions were obtained. Enzymatic treatment with papain or pronase and staining with an ethidiumbromide-mithramycin dye solution generate stoichiometric DNA-staining including that of mature sperm with a coefficient of variation below 2%.     

Assembly of mammalian septins

Septins are a conserved family of polymerizing guanine nucleotide binding proteins associated with diverse processes in dividing and non-dividing cells. In humans, 12 septin genes generate dozens of polypeptides, many of which comprise heterooligomeric complexes. Native and recombinant mammalian septin complexes are purified as approximately 8-nm-thick filaments of variable length. Ultrastructurally, a mammalian septin filament appears an irregular array of structural segments, whose polarity is obscure. The filaments have a potential to self-assemble into higher-order structures by lateral stacking and tandem annealing, eventually forming uniformly curved bundles, i.e., rings and coils. The septin filaments also undergo templated assembly along existing actin bundles containing an adapter protein, anillin. The resultant higher-order assembly of septin filaments may provide scaffolds to recruit other molecules and/or help organize the actin-based structures. The in vitro self-assembly is an irreversible process, which is not coupled with robust nucleotide exchange or hydrolysis. In contrast, septin-based structures rearrange and disassemble in cells, which might be controlled by diverse factors (e.g., the Cdc42-borg system, anillin, syntaxin, phospholipids) and covalent modifications (e.g., phosphorylation, ubiquitination, sumoylation). An immediate goal of septin biochemistry is to define the mechanisms of assembly and disassembly of this elusive cytoskeleton.     

Multifaceted mammalian transcriptome

Despite surprisingly a small number of protein-coding gene in mammalian genomes, a large variety of different RNAs is being produced. These RNAs are amazingly different in their number, size, cell localization, and mechanism of actions. Although new classes of short RNAs (sRNAs) are being continuously discovered, it is not yet obvious how many of the sRNAs are originated. Altogether, the research in the recent few years has identified an unexpectedly rich variety of mechanisms by which noncoding RNAs act, suggesting that we have identified probably only few of the many potential functional mechanism and more investigation will be needed to comprehensively understand the complex nature and biology of mammalian RNAome. Here, we focus on various aspects of the diversity of the biological role of these nonprotein-coding RNAs (ncRNAs), with emphasis on functional mechanisms recently elucidated.     

Purification methods of mammalian catechol-O-methyltransferases

The protein purification strategies used for obtaining homogeneous rat and human soluble catechol-O-methyltransferase (S-COTM) polypeptides are reviewed. Expression and purification of recombinant rat and human S-COMT in Escherichia coli and for human S-COMT in baculovirus-infected insect cells made it possible to elucidate the S-COMT polypeptides in more detail. The application of these purification methods has allowed the crystallization of the rat S-COMT protein and the analysis of the kinetic properties of the enzyme in great detail. The availability of the pure S-COMT protein together with the structural data has also greatly enhanced the development of more potent COMT inhibitors.