Metabolism of Round Spermatids: A Possible Regulation of Hexose Transport

Round spermatids (steps 1–8) were isolated from rat testes and glucose transport into the cells was examined. The exposure of spermatids to glucose resulted in an extremely low level of ATP. In contrast, the level of ATP remained constant in the presence of pyruvate. Transport of a glucose analogue, 2-deoxy-D-[³H]glucose ([³H]dGlc) into spermatids was correlated with intracellular levels of ATP and was much greater in cells with higher rather than lower levels of ATP. [³H]dGlc transport into spermatids with low levels of ATP was partially reversed when the cells were incubated with pyruvate. Inhibition of [³H]dGlc transport was exerted on Vmax and not on Km. Moreover, glucose acted as a competitive inhibitor of [³H]dGlc uptake (Km increased; Vmax unaltered). These results suggest that glucose transport into spermatids is active in vitro and probably regulated by the intracellular level of ATP.     

A new method in growth-electrophysiology: Pressurized intra-organ perfusion

Abstract A new experimental system was devised for the simultaneous measurement of elongation rate and the activity of the spatially separate electrogenic ion pumps of a hypocotyl segment excised from a seedling of Vigna unguiculata L. Walp. under enforced intra-organ perfusion by artificial solutions. The pathway of the perfusion medium was apoplastic space, including xylem vessels as main routes. The elongation rate of the segment was highly dependent on the perfusion pressure applied. It was possible to increase the growth rate under pressurized perfusion by 10-30 times as much as that without perfusion. Elongation rate was also dependent on respiration under perfusion, being retarded reversibly by anoxia a few minutes after the activities of the electrogenic ion pumps were stopped. Perfusion pressure had a little influence on the membrane potential (V_px) below a breakdown level (c. 130 kPa). Perfusion of mannitol or sorbitol solution of appropriate concentration reduced the elongation rate reversibly.     

Synergistic Inhibition of Fructose 1,6-Bisphosphatase by Fructose 2,6-Bisphosphate and Adenosine Monophosphate in Round Spermatids of Rats

The effects of adenosine monophosphate (AMP) and fructose 2, 6-bisphosphate (fruc-2, 6-P₂) on the key-enzyme of gluconeogenesis, fructose 1, 6-bisphosphatase (fruc-P₂ase; D-fructose 1, 6-bisphosphate 1-phosphohydrolase, EC 3.1.3.11) in spermatid extract from rat testes were studied. The fruc-P₂ase activity in the spermatids of rats was suppressed by AMP and fruc-2, 6-P₂. The inhibition of fruc-2, 6-P₂ was much stronger at low than at high substrate concentrations, and enhanced synergistically with AMP. The substrate saturation curve was changed by fruc-2, 6-P₂ hyperbolic to sigmoidal. Furthermore, the concentration of AMP that decreased the activity to 50% was much lower in the presence than in the absence of fruc-2, 6-P₂. These results indicate the possibility that gluconeogenesis in spermatids of rats is controlled by AMP and fruc-2, 6-P₂.     

THE SURFACE EVENTS AT FERTILIZATION OF THE SEA URCHIN EGG I. EVENTS ON THE SURFACE OF THE VITELLINE COAT1

Sperm-egg interaction during normal fertilization in the sea urchins, Strongylocentrotus intermedius and Hemicentrotus pulcherrimus, was studied by scanning and transmission electron microscopy. Several seconds after insemination, acrosome-reacted spermatozoa were found attached to the surface of the vitelline coat on each egg. Soon, several bulges of the vitelline coat appeared surrounding the fertilizing spermatozoon. These bulges then spread over the surface increasing in number, while they became fewer and disappeared around the sperm head. Thin sections of the bulging areas revealed discharging cortical granules. As the bulging vitelline coat was elevated, the sperm head was incorporated into the perivitelline space, passing through a small hole in the coat that resulted from penetration of the sperm acrosomal process immediately before fusion of the gametes. When the spermatozoon disappeared beneath the fertilization membrane, a hole was left in the membrane and the cortical reaction had finished on the other hemispheric surface. Mechanical removal of the membrane at that time exposed a spermatozoon protruding perpendicularly from the egg plasma membrane surface. The anterior tip of the sperm head was smoothly connected with the egg surface, and neither microvillous projections nor cytoplasmic covering of the egg cytoplasm could be found around the spermatozoon.     

The Effect of Oxygen on Melanin Precursors Released from Retinal Pigment Epithelial Cells In Vitro

The autoxidation of dopa to melanin in culture media causes toxicity to retinal pigment epithelial (RPE) cells and endothelial cells. The damage is specific to cell type and to the ambient oxygen concentration. To determine whether RPE cells influence the oxidation of dopa to media, we compared light absorbing dopa derivatives in the media exposed to cells with those found in the media incubated without cells. Dopa was extensively oxidized in the presence of RPE cells, and more light absorbing substances were generated with higher dopa and oxygen concentrations. However, an increase in ambient oxygen concentration decreased the quantity of several dopa derivatives which had been formed. The data provided evidence that RPE modulated dopa metabolism. Quinolic derivatives produced from a tyrosinase reaction and dopa-melanin formation moved the peak absorbance wavelength of dopa into the visible range. The spectrum between the dopa-derived compounds in the media has an absorbance at 240–275 nm and a maximum around 300 nm wth a shoulder near 375 nm. Gaussian analysis (peak separation) resolved these spectra into five components: a sharp band at 248 nm, a band at 295 nm, a large band at 359 nm, and two broad bands at 459 and 585 nm.     

CHANGES OF THE MEMBRANE POTENTIAL AT THE TIME OF FERTILIZATION IN THE SEA URCHIN EGG WITH SPECIAL REFERENCE TO THE FERTILIZATION-WAVE

An experimental device was developed from the work of U ehara and S ugiyama (1969), in order to study the electrical phenomena accompanying the fertilization-wave in the sea urchin egg.
The change in membrane potential upon fertilization consists of 2 peaks (I to et al. , 1970), being preceded by a shoulder. The shoulder appears within the "latent period" (A llen and G riffin , 1958), and the 2 peaks correspond to the breakdown of the cortical granules and the formation of the fertilization membrane.
When the equatorial region of the egg surface was exposed to a detergent-sea water, the breakdown of the cortical granules and the formation of the fertilization membrane are induced only in this ring-shaped area. Sperm is then added to one of the polar regions. The fertilization-wave, starting from the point of sperm-entry, propagates across the detergent-treated region, and the membrane is formed on the whole egg surface. During such an experiment, changes of the membrane potential in the detergent-treated region were measured. 1 to 3 sudden transient depolarizations appear, followed by a delayed small depolarization. It is presumed that the initial depolarization corresponds to the fertilization-wave. The pattern of the potential change at normal fertilization may be explained by complexity of the cortical change, and the initial depolarizing shoulder is considered to correspond to the fertilization-wave, which is isolated by the above-mentioned device.     

CYTOLOGICAL AND ULTRASTRUCTURAL STUDIES ON MUSCLE DIFFERENTIATION IN THE ASCIDIAN,PEROPHORA ORIENTALIS*,**

Muscle cell differentiation in the tail of the ascidian, Perophora orientalis, from early tail-bud embryos to swimming larvae, were studied cytologically and ultrastructurally. Myogenic cells did not form multinucleated myotubes, but remained as mononucleated cells. Nucleolar component increased prior to a marked increase in cytoplasmic RNA. Cytoplasmic RNA appeared first around nucleus and later concentrated in the peripheral cytoplasm. The fine filaments measuring 20–30 Å in their thin parts and 30–45 Å in their thick parts in diameter appeared initially, forming loose networks, in the peripheral cytoplasm where ribosome clusters had been concentrated. These filaments were tightly attached by particles of various size and density. These filaments tended to be arranged in parallel as they increased in their size. They seemed to be precursors of both actin and myosin filaments of formed myofibrils. Z band precursors were found as dense patches in association with loosely arranged myofilaments and consisted of particulate and filamentous materials. The myofibrils seemed to grow further by organizing free filaments into bundles and further by aligning bundles of myofilaments at both ends.     

Distribution of electric potential and ion transport in the hypocotyl of Vigna sesquipedalis I. Distribution of overall ion concentration and the role of hydrogen ion in generation of potential difference

Overall concentration of free inorganic ions distributes inthe hypocotyl of a bean seedling {Vigna sesquipedalis) at aconstant level (H⁺) or decreases monotonously from the cotyledonarynode towards the base (K⁺, Na⁺, Ca⁺⁺ and Mg⁺⁺, phosphate, NO₃^–).According to our theory, this is inconsistent with the distributionof electric potential having a definite minimum in the elongatingregion. The discrepancy can not be explained by regional variancein radial potential difference or histological differentiationin passive ionic permeability of the cell membrane. Short circuitcurrent observed through a hypocotyl segment corresponded toa net flux in ions of 10–24 pEq/cm².sec. It is questionable,however, whether this is due to active ion transport, whichcan be the source of electric potential difference, or is apassive flow due to histological heterogeneity in ion concentration. In order to investigate the latter possibility, pH of sap exudingfrom stumps made at various intervals along the hypocotyl axiswas measured, since H⁺ is the ion electro-osmotically most effective.pH Values of acropetal exudates distributed along the axis closelycorresponding to the distribution of electric potential. Thissuggests that potential distribution is determined by a passiveflow of H⁺ through a specific channel in the vascular system.The fact that H⁺ production and the uptake of ions and waterare most active at the elongating zone of hypocotyl is discussedfrom a physiological point of view. (Received December 3, 1969; )     

Melanin Granules Prevent the Cytotoxic Effects of l‐DOPA on Retinal Pigment Epithelial Cells in Vitro by Regulation of NO and Superoxide Radicals

Inasmuch as the nitrogen cycle elicits the direct reduction of N₂ to NH₃ through enzymatic reactions, and inasmuch as l ‐DOPA ( l ‐dihydroxyphentlalamine), a catecholamine, can be a source of nitric oxide (NO), it is possible that melanin granules in the eye affect the generation of NO, which causes damage to the retinal pigment epithelial (RPE) cells during the oxidation of l ‐DOPA. In order to confirm this possibility, we analyzed the correlations of NO generation, cell growth, and superoxide dismutase (SOD) activities in two types (melanotic and amelanotic) of bovine RPE cells following exposure to l ‐DOPA. NO generation from l ‐DOPA was determined using an NO detector that is reliant on redox currents. The concentration of NO was measured in terms of diffusion currents run between a working electrode and a counter electrode, both being set in culture medium placed in a Petri dish. For the assays, l ‐DOPA was added to the medium at various concentrations (5, 29.9, 79.4, 152.7 or 249 μM), and 6 min after addition, an NO‐trapping agent 2,4‐carboxyphenyl‐4,4,5,5‐tetramethylimidazole‐1‐oxyl 3‐oxide (carboxy‐PTIO) was also added. The melanotic and amelanotic types of RPE cells were cultured separately in medium with l ‐DOPA under an atmosphere containing 20, 10 or 5% oxygen. Cell numbers were counted using a Coulter counter, and SOD activities were determined following incubation for 24, 48 or 72 hr using a modification of the luminol assay. The results obtained indicated that: (a) NO was produced from l ‐DOPA in a concentration‐dependent manner and was trapped quantitatively by carboxy‐PTIO; (b) the generation of NO was inhibited more markedly in the melanotic cell line than in the amelanotic one, suggesting an increased tolerance to l ‐DOPA‐derived cytotoxicity in the former; and (c) the SOD activities were more affected by oxygen concentration in the melanotic cells than in the amelanotic ones. From these results, it is concluded that melanin granules in RPE cells have a role in preventing the cytotoxicity derived from l ‐DOPA and in regulating the generation of NO and superoxide radicals.