Detection of Y-bearing porcine spermatozoa by in situ hybridization using digoxigenin-labeled,porcine male-specific DNA probe produced by polymerase chain reaction

This study was carried out to determine whether Y-bearing porcine spermatozoa could be detected by in situ hybridization using a digoxigenin (Dig)-labelled DNA probe specific to the Y chromosome produced by polymerase chain reaction (PCR). A conventional PCR (with Dig-dUTP) was performed using a set of oligonucleotide primers (5′-AAGTGGTCAGCGTGTCCATA-3′ and 5′-TTTCTCCTGTATCCTCCTGC-3′) for 236 bp fragment of porcine male-specific DNA sequence and 1.25 × 10⁴ template white blood cells obtained from a boar. When fluorescence in situ hybridization with the Dig-labelled DNA probe was applied to the metaphase chromosome spreads prepared from both boar and gilts, the fluorescein signal was only detected on the long arm of the Y chromosome. In addition, immunocytochemical detection with the Dig-labelled DNA probe and alkaline phosphatase-labeled anti-Dig was applied to both sperm nuclei pretreated with dithiothreitol and white blood cells; 51% of sperm nuclei and 96% of white blood cells obtained from boar were labelled, whereas none of white blood cells obtained from gilts were labelled with the Dig-labelled DNA probe. The results indicated that in situ hybridization with porcine male-specific DNA probe produced by PCR made possible the direct visualization of Y-bearing porcine spermatozoa by in situ hybridization. © 1995 Wiley-Liss, Inc.     

Bovine cumulus cell expansion does not depend on the presence of an oocyte secreted factor

Communication between the oocyte and its somatic cells has been shown to be important in oocyte development. Here we examined how the oocyte may be involved in bovine cumulus cell expansion. Intact bovine cumulus oocyte complexes (COC) were obtained by puncturing antral follicles. From the intact COC, oocytectomised complexes (OOX) were produced by micro surgical removal of the oocyte. Clumps of cumulus cells (CC) were obtained by micro-dissection. Intact or OOX complexes or CC were matured in the presence of fetal calf serum and hFSH (6 mlU/ml) for 24 hr and the degree of expansion measured. The presence of the oocyte is not essential to allow bovine cumulus expansion to occur as expansion occurred in all groups. Murine OOX complexes from eCG primed 35–40-day-old C57BL6/CBA F₁ hybrids (known to require the presence of an oocyte secreted factor for cumulus expansion) were cultured with or without denuded bovine oocytes (1 oocyte/μl). Murine OOX complexes expanded only in the presence of denuded bovine oocytes. Thus some factor produced by bovine oocytes enabled expansion of murine OOX complexes. To determine whether the factor is secreted by bovine oocytes, murine OOX were cultured with or without media conditioned by bovine oocytes (1 oocyte/μl for 4 hr). Significant expansion of murine OOX occurred in media conditioned by bovine oocytes. This shows that the cumulus expansion enabling effect of bovine oocytes is released into the surrounding media. Media conditioned by bovine oocytes and then frozen for up to 1 month showed that the activity by the factor can withstand freezing. © 1995 wiley-Liss, Inc.     

Mechanism of toxicity of precocene II in rat hepatocyte cultures

Precocene II was more toxic in 24 hour cultures than in 72 hour cultures of rat hepatocytes. In 24 hour cultures, there was no observable toxicity at 75 μM precocene II after exposure for 6 hours, but after 24 hours, 65% of the cells were dead. In contrast, although 794 μM killed 50% of the cells in the 72 hour cultures after a 24 hour exposure, 1 mM killed 96% of the cells within 6 hours. In both 24 and 72 hour cultures, cell death was preceded by a rapid, early loss of mitochondrial membrane potential, followed by decreases in glutathione, reduced pyridine nucleotide status, and plasma membrane Na⁺/K⁺-ATPase activity. There was also a rapid loss of ATP in the 72 hour cultures but not in the 24 hour cultures; therefore, onset of cell death may be closely linked to loss of ATP. Inhibition of cytochrome P-450 prevented the toxicity, and partially protected against the loss of membrane potential and glutathione, in 24 hour cultures but was ineffective in 72 hour cultures. Therefore, in addition to depletion of glutathione, precocene II appears to damage mitochondria and plasma membrane functions and can do so by more than one pathway. © 1996 John Wiley & Sons, Inc.     

Changes in Metabotropic Glutamate Receptor mRNA Levels Following Global Ischemia: Increase of a Putative Presynaptic Subtype (mGluR4) in Highly Vulnerable Rat Brain Areas

Abstract: Metabotropic glutamate receptors mediate their intracellular response by coupling to G proteins and may be divided into three subfamilies: mGluR1 and mGluR5, which stimulate phosphatidylinositol hydrolysis; mGluR2 and mGluR3, which are negatively coupled to cyclic AMP formation; and mGluR4 and mGluR6, which also inhibit forskolin-stimulated cyclic AMP formation. The mGluR4 subtypes may represent l -2-amino-4-phosphonobutyrate-sensitive presynaptic autoreceptors, and two alternatively spliced variants of the mGluR4 coding for two receptors with different C termini have been identified. Using in situ hybridization, we measured the levels of mGluR1–mGluR5 mRNA in regions of the rat brain 24 h after transient global ischemia, a time point when no neuronal damage can yet be observed morphologically. In the hippocampus, the mRNA levels for mGluR1, mGluR2, and mGluR5 were decreased, mGluR3 mRNA levels were unchanged, and the mGluR4 mRNA levels were strongly increased. The strongest increase appeared to be in the mRNA encoding mGluR4b. The mGluR4 mRNA was also increased in the parietal cortex, whereas the ventral posteromedial thalamic nucleus showed a small decrease in its mRNA content. These results suggest that vulnerable neurons react to an increased extracellular glutamate concentration by differential regulation of the mRNA for pre- and postsynaptically located metabotropic glutamate receptors.     

Regulation of μ-Opioid Receptor mRNA in Rat Globus Pallidus: Effects of Enkephalin Increases Induced by Short- and Long-Term Haloperidol Administration

Abstract: The mRNA encoding μ-opioid receptors is expressed in neurons of the globus pallidus, a region of the basal ganglia that receives a dense enkephalinergic innervation from the striatum. The regulation of the mRNAs encoding the opioid peptide enkephalin in the striatum and the μ-opioid receptor in the globus pallidus was examined with in situ hybridization histochemistry following short- or long-term haloperidol treatments, which alter striatal enkephalin mRNA levels. Animals were administered haloperidol daily for 3 or 7 days (1 mg/kg, s.c.) or continuously for 8 months (1 mg/kg, depot followed by oral). Enkephalin and μ-opioid receptor mRNA levels were unchanged after 3 days of haloperidol treatment. In contrast, the enkephalin mRNA level was increased in the striatum, and μ-opioid receptor mRNA levels were markedly decreased in the globus pallidus after 7 days of haloperidol administration. Similar effects were observed in rats treated with haloperidol for 8 months. The results provide the first evidence of regulation of μ-opioid receptor mRNA in vivo.     

Chromosome painting of Amigo wheat

Chromosome painting using multicolor fluorescence in situ hybridization showed that, in addition to the T1AL·1RS translocation derived from rye, a segment from chromosome 3Ae#1 of Agropyron elongatum (2n=10x =70), is present in Amigo wheat. The Agropyron chromosome segment is located on the satellite of chromosome 1B and the translocation chromosome is designated as T1BL·1BS-3Ae#1L. T1BL·1BS-3Ae#1L was inherited from Teewon wheat and carries resistance genes to stem rust (Sr24) and leaf rust (Lr24). The Agropyron chromosome segments in different Sr24/Lr24 carrier wheat lines, including Agent, TAP 48, TAP 67, Teewon, and Amigo, showed a diagnostic C-band, and were derived from the same chromosome, 3Ae#1.     

A genetic approach to the identification of functional amino acids in protein p6 of Bacillus subtilis phage ?29

Protein p6 of the Bacillus subtilis phage ø29 is essential for in vivo viral DNA replication. This protein activates the initiation of ø29 DNA replication in vitro by forming a multimeric nucleoprotein complex at the replication origins. The N-terminal region of protein p6 is involved in DNA binding, as shown by in vitro studies with p6 proteins altered by deletions or missense mutations. We report on the development of an in vivo functional assay for protein p6. This assay is based on the ability of protein p6-producing B. subtilis non-suppressor (su ^–) cells to support growth of a ø29 sus6 mutant phage. We have used this trans-complementation assay to investigate the effect on in vivo viral DNA synthesis of missense mutations introduced into the protein p6 N-terminal region. The alteration of lysine to alanine at position 2 resulted in a partially functional protein, whereas the replacement of arginine by alanine at position 6 gave rise to an inactive protein. These results indicate that arginine at position 6 is critical for the in vivo activity of protein p6. Our complementation system provides a useful genetic approach for the identification of functionally important amino acids in protein p6.