Convoluted cords, a new nuclear body in spermatocytes and spermatids of the rabbit

A voltammetric study of the oxidation of terbutaline has been carried out at an activated glassy carbon electrode. The compound was oxidized irreversibly at high positive potential. The response was evaluated with respect to pH, scan rate, nature of the buffer and other variables. The peak current, at about 0.8 V (versus a saturated calomel electrode), was proportional to the terbutaline concentration in the range of 8 x 10(-6)-8 x 10(-4) M in phosphate buffer pH 6.0. This method was applied, without any interferences from the excipients, to determine the drug in a tablet dosage form.     

Cyclin synthesis controls the progression of meiotic maturation in mouse oocytes

To study the mechanisms involved in the progression of meiotic maturation in the mouse, we used oocytes from two strains of mice, CBA/Kw and KE, which differ greatly in the rate at which they undergo meiotic maturation. CBA/Kw oocytes extrude the first polar body about 7 hours after breakdown of the germinal vesicle (GVBD), whilst the oocytes from KE mice take approximately 3-4 hours longer. In both strains, the kinetics of spindle formation are comparable. While the kinetics of MAP kinase activity are very similar in both strains (although slightly faster in CBA/Kw), the rise of cdc2 kinase activity is very rapid in CBA/Kw oocytes and slow and diphasic in KE oocytes. When protein synthesis is inhibited, the activity of the cdc2 kinase starts to rise but arrests shortly after GVBD with a slightly higher level in CBA/Kw oocytes, which may correspond to the presence of a larger pool of cyclin B1 in prophase CBA/Kw oocytes. After GVBD, the rate of cyclin B1 synthesis is higher in CBA/Kw than in KE oocytes, whilst the overall level of protein synthesis and the amount of messenger RNA coding for cyclin B1 are identical in oocytes from both strains. The injection of cyclin B1 messenger RNA in KE oocytes increased the H1 kinase activity and sped up first polar body extrusion. Finally, analysis of the rate of maturation in hybrids obtained after fusion of nuclear and cytoplasmic fragments of oocytes from both strains suggests that both the germinal vesicle and the cytoplasm contain factor(s) influencing the length of the first meiotic M phase. These results demonstrate that the rate of cyclin B1 synthesis controls the length of the first meiotic M phase and that a nuclear factor able to speed up cyclin B synthesis is present in CBA/Kw oocytes.     

RNA synthesis in preovulatory mouse oocytes

RNA synthesis, previously shown to take place during oocyte growth, has been demonstrated throughout the growth-quiescent period preceding ovulation of the mouse oocyte. In the final 7-day preovulatory period, the level of incorporation of (5,6-3H)uridine into ovulated oocytes decreased as the interval between exposure to precursor and ovulation decreased; significant incorporation was detectable within 2 days before ovulation. Analysis of the frequency and density of label in ovarian oocytes at successive stages of meiosis in relation to the interval between adminstration of labeled precursor and collection of oocytes revealed that RNA synthesis continues up to within 2 h before GVBD.     

Thermosensitivity of nuclear RNA polymerase during the in vitro senescence of mouse fibroblasts

Embryonic mouse fibroblasts divide approximately twelve times in vitro prior to cessation of mitotic activity. During this period of cellular senescence the thermosensitivity of the RNA polymerase activity of isolated nuclei has been examined as a means of detecting the possible accumulation of defective enzyme molecules, as has been found by other workers for several cytoplasmic enzymes during the ageing of human fibroblasts in vitro. The total RNA polymerase activity of nuclei isolated from old (10th generation) cells is more thermoresistant than that of young (2nd generation) cells. However, the net RNA polymerase activity of nuclei from non-dividing (confluent) cells is more thermoresistant than that of exponentially growing cells of the same age. When allowance is made for the state of growth of the cultures, little difference is seens in the thermosensitivity of the activities of nuclei from old and young cells. Neither is there any difference between the thermosensitivity of the net activity of an established line of murine fibroblasts (L-cells) and cells in primary culture. Preheating nuclei increases the inhibition of their total RNA polymerase activity by alpha-amanitin, indicating that RNA polymerase II is the most heat resistance species present. There appears to be no difference between the thermosensitivity of the alpha-amanitin sensitive and resistance species of the enzyme in the nuclei of old and young cells. It is concluded that old cells resemble non-dividing young cells in containing a higher proportion of RNA polymerase II in their nuclei, resulting in greater thermoresistance of the total RNA polymerase activity over that of exponentially growing cells. However, there appears to be no increase in thermosensitivity of the enzymes with age.     

Stability of RNA synthesized by the mouse oocyte during its major growth phase

Follow-up examinations of 82 children after surgery for subdjral effusions show a varied degree of retardation of motor- and speech-development. This corresponds to neurologic signs of varying severity. Preoperative EEG findings are improved both in pre-school- and school children. Disturbance of motor and intellectual development as a rule corresponds to severe EEG changes. Among pre-school children 35% had an IQ below 69%, among school-children 36%. But their occupational chances cannot yet be finally assessed. Children with large effusions (over 50 ml) and extensive capsules show a higher percentage of retardation. Present results do not establish relations between site of effusion, frequency of recurrence and mental development.     

A novel stage-specific differentiation antigen is expressed on mouse testicular germ cells during early meiotic prophase

A murine cell surface antigen exhibiting stage-specific expression during spermatogenesis was detected with two monoclonal antibodies (mAbs), designated BC7 and CA12. In mouse testis, these mAbs recognized a small population of cells located near the periphery of seminiferous tubules at stages XII and I-VI, and these spermatogenic cells were identified as zygotene and early pachytene spermatocytes. Expression of the antigens was transient and was not detected in germ cells at more advanced stages of spermatogenesis such as late pachytene spermatocytes and round spermatids. Immunoprecipitation and immunoblotting studies showed that both mAbs CA12 and BC7 reacted with the same antigenic molecule, which had an estimated molecular mass of 95 kDa. CA12/BC7 antigen, detected in plasma membrane fraction, was a glycoprotein with sialic acid residues and had affinity with WGA lectin. Furthermore, intraperitoneal injection of mAb BC7 caused an apparent spermatogenic disturbance in prepubertal mice. These results suggested that CA12/BC7 antigen, a novel cell surface glycoprotein, is an essential molecule that plays an important role during early meiotic prophase of spermatogenesis.     

Angiotensin-converting enzyme in murine testis: step-specific expression of the germinal isoform during spermiogenesis

Angiotensin I-converting enzyme (ACE) is known primarily as an endothelial enzyme that plays a critical role in the regulation of blood pressure. Another, shorter isoform of ACE is abundantly expressed in the testes of sexually mature animals. Using antibodies for immunoperoxidase detection and [35S]-labeled riboprobes for in situ hybridization (ISH), we studied the temporal expression and cell distribution of this germinal isoform of ACE in the testis of normal mice and rats as well as of pubertal and sterile mice. In both murine species, specific testicular ACE mRNA and its gene product are present only after completion of meiosis. Through studying two murine species in which spermatogenesis and spermiogenesis have been accurately described, as well as immature and sterile animals, it could be shown that ACE mRNA and its corresponding protein are first synthesized during the cap phase (steps 4-7). The maximum expression occurred during the acrosome phase (steps 8-12). ACE mRNA is no longer detectable in spermatids beyond step 14, whereas its gene product is expressed until the end of spermatid maturation. Therefore, ACE is exclusively produced in haploid germ cells and belongs to the growing family of proteins whose expression during definite maturation steps of spermiogenesis appears to be correlated with the unique process of germ cell differentiation.     

The Sycp1 loci of the mouse genome: successive retropositions of a meiotic gene during the recent evolution of the genus

We wished to determine the effect of altering the levels or functional activity of retinoid receptors, in particular retinoic acid receptor-alpha (RAR-alpha) and retinoid X receptor-alpha (RXR-alpha) on the growth sensitivity of ovarian tumor cells to all-trans-retinoic acid (all-trans-RA). We found that CA-OV3 cells could be made resistant to all-trans-RA growth inhibition by overexpressing RAR-beta(R269Q), an efficient dominant negative mutant which inhibits the function of all RAR subtypes. Antisense technology was then used to prepare stable transfectants of the retinoid-sensitive ovarian carcinoma cell line CA-OV3 in which expression of RAR-alpha, RXR-alpha, or both RAR-alpha and RXR-alpha was reduced. The effect of all-trans-RA on ovarian tumor cell growth was determined by MTT assay, autoradiographic analysis of DNA synthesis, and anchorage-independent colony formation in soft agar. Our results show that cell lines expressing reduced levels of either RAR-alpha alone or RXR-alpha alone exhibited a small decrease in sensitivity to growth inhibition by all-trans-RA. However, maximum RA resistance was obtained in cell lines in which the levels of both RAR-alpha and RXR-alpha were reduced. These results demonstrate the importance of both retinoid nuclear receptors and retinoid-X receptors in general, and RAR-alpha and RXR-alpha in particular, as mediators of ovarian carcinoma cell growth inhibition by retinoids.     

Genotoxicity of trophosphamide in mouse germ cells: assessment of micronuclei in spermatids and chromosome aberrations in one-cell zygotes

We have used the muscle history dependence of the sensitivity of muscle spindles to stretch, to provide evidence for their contribution to kinaesthesia, the sense of position and movement. Stretch sensitivity is altered depending on whether or not slack has been introduced in intrafusal fibres [13]. At the human elbow joint detection threshold was measured to passive movements applied at different speeds to the forearm after a conditioning co-contraction of muscles of the upper arm, with the arm held either flexed ('hold short') or extended ('hold long'). Test measurements were made with the elbow joint at 90 degrees. For the three speeds of movement, 2 degrees s-1, 0.2 degree s-1 and 0.02 degree s-1, after 'hold short' conditioning thresholds were lower for movements into extension, after 'hold long' conditioning they were lower for movements into flexion. It is concluded that when muscle conditioning introduces slack in the intrafusal fibres of muscle spindles, this must be taken up by the test movements before they can be detected by the subject. It means that whenever detection thresholds to passive movements are measured at a joint, the contraction history of the muscles acting at that joint must be taken into account.     

Differential expression and functions of cortical myosin IIA and IIB isotypes during meiotic maturation, fertilization, and mitosis in mouse oocytes and embryos

To explore the role of nonmuscle myosin II isoforms during mouse gametogenesis, fertilization, and early development, localization and microinjection studies were performed using monospecific antibodies to myosin IIA and IIB isotypes. Each myosin II antibody recognizes a 205-kDa protein in oocytes, but not mature sperm. Myosin IIA and IIB demonstrate differential expression during meiotic maturation and following fertilization: only the IIA isoform detects metaphase spindles or accumulates in the mitotic cleavage furrow. In the unfertilized oocyte, both myosin isoforms are polarized in the cortex directly overlying the metaphase-arrested second meiotic spindle. Cortical polarization is altered after spindle disassembly with Colcemid: the scattered meiotic chromosomes initiate myosin IIA and microfilament assemble in the vicinity of each chromosome mass. During sperm incorporation, both myosin II isotypes concentrate in the second polar body cleavage furrow and the sperm incorporation cone. In functional experiments, the microinjection of myosin IIA antibody disrupts meiotic maturation to metaphase II arrest, probably through depletion of spindle-associated myosin IIA protein and antibody binding to chromosome surfaces. Conversely, the microinjection of myosin IIB antibody blocks microfilament-directed chromosome scattering in Colcemid-treated mature oocytes, suggesting a role in mediating chromosome-cortical actomyosin interactions. Neither myosin II antibody, alone or coinjected, blocks second polar body formation, in vitro fertilization, or cytokinesis. Finally, microinjection of a nonphosphorylatable 20-kDa regulatory myosin light chain specifically blocks sperm incorporation cone disassembly and impedes cell cycle progression, suggesting that interference with myosin II phosphorylation influences fertilization. Thus, conventional myosins break cortical symmetry in oocytes by participating in eccentric meiotic spindle positioning, sperm incorporation cone dynamics, and cytokinesis. Although murine sperm do not express myosin II, different myosin II isotypes may have distinct roles during early embryonic development.     

Telomere length, telomerase activity and telomerase RNA expression during mouse mammary tumor progression

The effects of intraportal administration of prostaglandin E1 (PGE1) on portal venous flow, hepatic arterial flow, peripheral tissue blood flow, and systemic arterial flow before and after 60 min total liver ischemia followed by 70% partial hepatectomy in rats were investigated. Total liver ischemia was induced by occluding the hepatoduodenal ligament for 60 min. PGE1 at a dose of 0.5 microg/kg/min was infused intraportally for 15 min before inducing hepatic ischemia (preischemic period) and for 60 min after ischemia (postischemic reperfusion period) in the treatment group. Normal saline was infused in the control group. Seventy percent partial hepatectomy was performed during ischemia. Serum biochemical analysis and liver tissue histology were carried out 1, 3, and 24 h, and 1 and 24 h after reperfusion respectively. One-week survival of the PGE1 group was improved to 70% compared to that of the control group of 30%. Postischemia reperfusion values of portal and peripheral tissue blood flows in the PGE1 group were 6.33 +/- 0.600 ml/min and 27.2 +/- 23.5 (arbitrary), and were significantly different from those of the control group of 4.34 +/- 0.400 ml/min and 23.5 +/- 5.54 (arbitrary), respectively. There was no significant difference in hepatic arterial flow between the two groups. Serum alkaline phosphatase decreased significantly in the prostaglandin group. Histological examination revealed a significant portal venous congestion in the control group 1 and 24 h after reperfusion. The extent of the sinusoidal congestion was also severe in the control group 24 h after reperfusion. It was concluded that PGE1 has a protective effect against liver damage when the liver was injured by warm ischemia and reperfusion followed by partial resection.     

Structural changes in the endoplasmic reticulum of starfish oocytes during meiotic maturation and fertilization

The endoplasmic reticulum (ER) of live starfish oocytes was observed during meiotic maturation and fertilization. The ER was visualized by injection into the cytoplasm of an oil drop saturated with the fluorescent lipophilic dye DiI; DiI spread throughout the oocyte endoplasmic reticulum and the pattern was imaged by confocal microscopy. The ER in the immature (germinal vesicle stage) oocyte was composed of interconnected membrane sheets. In response to 1-methyladenine, the sheets of ER appeared to become associated with the yolk platelets, forming spherical shells. A few of these spherical shells could sometimes be seen in immature oocytes, but their number was much greater in the egg at the first meiotic spindle stage. At about the time that the first polar body formed, the spherical shells disappeared, and the ER returned to a form like that of the immature oocyte. The spherical shells did not reappear during the second meiotic cycle. During maturation, the ER also began to move; the movement was apparent by the time of germinal vesicle breakdown and continued throughout both meiotic cycles and in eggs with second polar bodies. When eggs at the first meiotic spindle stage were fertilized, the form of the ER changed. Within 1 min after sperm addition to the observation chamber, the circular cross sections of the spherical shells of the unfertilized egg ER were no longer distinct. At this point, the form of the ER could not be discerned with the resolution of the light microscope; however, the rate of spreading of DiI from an injected oil drop decreased, providing strong evidence that the ER had become fragmented. The ER remained in this form for several minutes and then gradually, the appearance of the ER and the rate of DiI spreading returned to be like those of the unfertilized egg. Injection of inositol trisphosphate caused a similar change in the ER structure. These results indicate that the ER is a dynamic structure, the form of which changes during oocyte maturation and fertilization.     

Calcium/calmodulin-dependent protein kinase II and calmodulin: regulators of the meiotic spindle in mouse eggs

Elevation of intracellular free calcium causes egg activation by initiating a cascade of interacting signaling pathways that, in unison, act to remodel the cytoplasmic compartment and the nuclear compartment of the egg. We show here that calcium/calmodulin-dependent protein kinase II (CaM kinase II) is tightly associated with the meiotic spindle and that 5 min after egg activation there is a transient, tight association of calmodulin (colocalized with CaM kinase II) on the meiotic spindle. These correlative observations caused us to test whether activation of CaM kinase II mediated the chromosomal transit into an anaphase configuration. We demonstrate that calcium and calmodulin, at physiological levels, along with ATP were capable of driving the spindle (with its associated CaM kinase II) into an anaphase configuration in a permeabilized egg system. The transit into anaphase was dependent on the presence of both calcium and calmodulin and occurred normally when they were present at a ratio of 4 to 1. Peptide and pharmacologic inhibitors of CaM kinase II blocked the transit into anaphase, both in the permeabilized egg system and in living eggs (inhibitors of protein kinase C did not block the transit into anaphase). Using a biochemical approach we confirm that CaM kinase II increases in activity 5 min after egg activation and that a second increase occurs 45 min after activation at the approximate time that the contractile ring of the second polar body is constricting. This corresponds to the approximate time when calmodulin and CaM kinase II colocalize at several points in the activated egg including the region containing midzone microtubules. CaM kinase II appears localized on midzone microtubules as soon as they form and may have a role in specifying the position of the contractile ring of the second polar body.