Feeding rates and absorption efficiencies of four species of sea urchins (genus Echinometra) fed a prepared diet

Four closely related species of sea urchins belonging to the genus Echinometra, Echinometra sp. A (Ea), E. mathaei (Em), E. sp. C (Ec), and E. oblonga (Eo), occur sympatrically but in different microhabitats on Okinawan coral reefs. Feeding rates and absorption efficiencies of the four species were investigated in the laboratory by feeding sea urchins ad libitum a diet prepared from turf algae and agar over a 7-day period. Feeding rates differed significantly among the four species of Echinometra (Ea > Em approximately = Ec > Eo). Absorption efficiencies of protein and lipid did not differ significantly among the four species. Carbohydrate, a major nutrient component in the diet, was absorbed by the four species at significantly different efficiencies (Eo > Ec approximately = Em > Ea), which resulted in similar interspecific differences in absorption efficiencies of dry matter, total organic matter, and energy. The amount of nutrients absorbed from the diet was directly related to the feeding rate, indicating that the increase in absorption efficiency was not sufficient to completely compensate for low feeding rate. The interspecific difference in physiological performance in relation to feeding and absorption is consistent with taxonomic differentiation among the four species.     

Hybrids of two closely related tropical sea urchins (genus Echinometra): evidence against postzygotic isolating mechanisms

A series of cross-fertilization experiments were conducted with two unnamed, sympatric species of sea urchins in the Echinometra mathaei species complex, Echinometra sp. A (Ea) and Echinometra sp. C (Ec). Heterogametic fertilization success was high when eggs of Ec and sperm of Ea were involved, and low with eggs of Ea and sperm of Ec. Hybrids produced from crosses in either direction developed normally to sexually mature adults; Ea x Ea were largest in test size, followed by Ec (ova) x Ea (sperm), Ea (ova) x Ec (sperm), and Ec x Ec, respectively. Color patterns of the hybrids were closer to the maternal coloration, whereas other characters such as relative test dimensions and spine lengths, morphology of tubefoot and gonad spicules, and gamete sizes were intermediate. Fertilization rates in F1 backcrosses were high, minimizing the possibility that hybrid infertility is a postzygotic mechanism of reproductive isolation. On the other hand, intensive surveys failed to find individuals with hybrid characteristics in the field, suggesting that natural hybridization between the two species is rare. Prezygotic isolating mechanisms, such as microhabitat separation and gamete incompatibility, at least between Ea eggs and Ec sperm, most likely maintain the genetic integrity of these two closely related species.     

Hybridization and F1 backcrosses between two closely related tropical species of sea urchins (genus Echinometra) in Okinawa

Summary

Four species of sea urchins of the genus Echinometra, designated species A, B, C, and D, occur along the coast of Okinawa; they are distinguished by color pattern and other characteristics. The two most distinct species, Echinometra sp. A (Ea) with white-tipped spines and Echinometra sp. D (Ed) with nearly black spines (possibly E. oblonga), were examined for potential hybridization through a series of fertilization experiments and rearing of the resulting hybrids. Fertilization was reciprocally asymmetrical; Ed ova were readily fertilized by sperm of Ea, but less than 20% of the ova of Ea were fertilized by sperm of Ed. Nevertheless, hybrids resulting from crosses in both directions developed normally through larval and juvenile stages to produce sexually mature adults. Larvae of Ed♀×Ea♂ and Ed♂×Ed♀ were larger than those of Ea♂ × Ed♀ and Ea♂ × Ea♀, reflecting the larger eggs of Ed. However, Ea♂×Ea♀ juveniles and later stages, up to maturity, were consistently larger than those of equivalent ages of the other crosses; largest to smallest were Ea♂×Ea♀, Ed♂×Ea♀, Ea♂ × Ed♀, and Ed♂×Ed♀, respectively. Ova and sperm of the hybrids were viable and fertilizable in all combination of crosses, although fertilization of those of Ed♂×Ea♀ hybrids was consistently higher than those of Ea♂ × Ed♀ hybrids. These experiments represent the first reported successful production of hybrid sea urchins with viable gametes. They indicate that reproductive isolation is achieved by prezygotic isolating mechanisms in these two species. Of these mechanisms, gametic incompatibility is probably only partly involved, if at all, and differences in spawning times, habitat segregation, or other factors appear to be important for maintaining reproductive isolation in these closely related species.     

Energy metabolism of sea urchin spermatozoa, with phosphatidylcholine as the preferred substrate

Phosphatidylcholine content in the spermatozoa of the sea urchin, Hemicentrotus pulcherrimus, decreased rapidly during incubation with sea water. Sea urchin sperm contained approx. 85% phospholipid in total lipid. Phosphatidylcholine (PC) was the principal lipid. Other phospholipids, however, remained at constant levels during incubation. Although the free fatty acid content gradually increased following dilution of dry sperm in sea water, the amounts of triacylglycerol and cholesterol ester were extremely low. Analysis by gas-liquid chromatography indicated most of fatty acid moieties in PC to be polyenoic. PC composed in part of unsaturated fatty acids was consumed to a greater extent during incubation than that consisting of saturated fatty acids. Furthermore, 1-palmitoyl-2-[1-14C]linoleoylphosphatidylcholine was transformed to 14C-labelled free fatty acid in a subcellular system. Thus, possibly, phospholipase A2 is present in sea urchin sperm. Also, [1-14C]oleic acid was immediately oxidized to 14CO2 by sperm. It is thus concluded that sea urchin sperm use phosphatidylcholine as a substrate for energy metabolism.     

Localization and characterization of phosphatidylcholine in sea urchin spermatozoa.

Sea urchin spermatozoa obtain energy for movement through oxidation of endogenous phospholipids, particularly phosphatidylcholine (PC). This study was undertaken to determine the localization of PC available for utilization in energy metabolism in spermatozoa of the sea urchin, Hemicentrotus pulcherrimus. Following incubation with sea water, the PC content in sperm heads decreased significantly, while that in sperm tails did not change. PC was abundant in sperm heads, particularly the midpieces. PC composed of unsaturated fatty acids was consumed to a greater extent during incubation than that consisting of saturated fatty acids. Analysis by gas-liquid chromatography indicated most of fatty acid moieties in the midpieces PC to be unsaturated. Phospholipase A2 activity was also distributed in sperm heads, particularly the midpieces. It thus appears that PC as a substrate for energy metabolism is located in the midpieces of sea urchin spermatozoa.     

意大利蜜蜂产浆性状10个微卫星位点的遗传分析

我国培育成功的世界上蜂王浆产量最高的蜂种（Ea）是从20世纪30年代引进我国的意大利蜜蜂（Eb）中选育的。采用10个微卫星位点对蜂王浆高产蜜蜂、原种意大利蜜蜂（Ee）和本地意大利蜜蜂进行研究,以探明由于人工选择和地理隔离造成的其分子进化上的一些特征。结果,10个微卫星位点在3个蜂种中共扩增到96个等位基因,其中有48个等位基因是不同的,表明10个微卫星位点在3个蜂种中的高度多态性,而且由于人工选择和地理隔离已造成3蜂种在遗传基础上的一些分化。3蜂种的多态信息含量（PIC）分别为0．57、0．50、0．57,杂合度分别为0．60、0．57、0．61,二者均无显著差异。遗传距离的分析结果显示,Ee和Eb（0．14）、Eb和Ea（0．16）之间的遗传距离较近,而Ea和Ee（0.25）之间的遗传距离较远。等位基因频率的分析结果表明,6个位点的7个等位基因的频率（A29的159bp、A24的100bp和104bp、A7的110bp、A43的126bp、A14的221bp和A113的221bp）以Ee、Eb、Ea的顺序递增,Ea的这7个等位基因频率分别显著高于Ee和Eb。同时4个位点的4个等位基因的频率（A24的106bp、A43的140bp;A113的215bp和A14的219bp）以Ea、Eb、Ee的Ⅲ页序下降,Ea在这4个位点的频率分别显著低于Eb和Ee。这些位点的等位基因可能与蜂王浆产量有关。     

Lipid globules at the midpieces of Glyptocidaris crenularis spermatozoa and their relation to energy metabolism

Spermatozoa of the sea urchin, Glyptocidaris crenularis, use endogenous triglycerides (TG), not phosphatidylcholine, to produce energy for swimming. This study was undertaken to examine ultrastructurally the location of TG available for utilization in energy metabolism in these spermatozoa. Each spermatozoon contained several lipid globules at the bottom of the midpiece. Following incubation with sea water, both the number and size of the lipid globules decreased significantly. The total volume of lipid globules in each spermatozoon was roughly halved after 1 h of incubation. Similarly, about half of the TG was metabolized during the same incubation period. Oxygen consumption by spermatozoa during the incubation indicated the oxidation of fatty acid derived from TG. Thus it appears that G. crenularis spermatozoa obtain energy through oxidation of fatty acid from TG stored in the lipid globules within their midpieces. © 1993 Wiley-Liss, Inc.     

Diacylglycerol species as messengers and substrates for phosphatidylcholine re-synthesis during Ca2+-dependent exocytosis in boar spermatozoa

We have investigated pathways of lipid metabolism in spermatozoa and generation of various metabolites with potential messenger functions during exocytosis stimulated with A23187/Ca²⁺. Stimulation of boar spermatozoa resulted in a considerable rapid increase in saturated/unsaturated 1,2-diacylglycerol (1,2-SU-DAG) and, concomitantly, a substantial reduction in disaturated 1,2-diacylglycerol (1,2-DS-DAG), and in phosphatidylcholine (PC). These changes preceded the onset of exocytosis. Phosphatidic acid was sometimes generated in parallel, but usually rose later, suggesting that 1,2-SU-DAG may be formed directly by phospholipase C action. Lipid changes observed in stimulated spermatozoa that have been prelabelled with several lipid precursors ([¹⁴C]palmitic acid, [¹⁴C]glycerol, [¹⁴C]choline, or [¹⁴C]arachidonic acid) suggested the existence of a unique process involving the utilization of the high basal levels of 1,2-DS-DAG to form 1,2-SU-DAG, with the latter being subsequently employed to replenish the PC pool. An ensuing generation of lysoPC and arachidonic acid, which paralleled the occurrence of exocytosis, revealed that the newly synthesized PC was hydrolyzed by phospholipase A₂. The highest levels of 1,2-SU-DAG, minimum levels of 1,2-DS-DAG, and the regeneration of the PC pool were tightly coupled to the beginning of visible exocytosis. These results suggest that changes in these lipid metabolites may be fundamental processes during acrosomal exocytosis occurring in response to physiological agonists. Mol. Reprod. Dev. 48:95–105, 1997. © 1997 Wiley-Liss, Inc.     

Alpha-tocopherol improves biochemical and dynamic parameters in cryopreserved boar semen

Cryopreservation is associated with the production of reactive oxygen species which lead to lipid peroxidation of sperm membranes. The objective was to determine an alpha-tocopherol concentration capable of improving the quality of cryopreserved porcine semen. Boar spermatozoa frozen with 200, 500 or 1000 microg/mL alpha-tocopherol were thawed and incubated at 37 degrees C for 4 h. Routine parameters of semen quality, susceptibility to lipid peroxidation 2-thiobarbituric acid (TBARS) and oxygen uptake were evaluated. Motility was higher (P<0.05) in samples treated with different concentrations of alpha-tocopherol up to 2 h of incubation. Viability and acrosome integrity significantly decreased during incubation (no significant differences between treatments). Two hundred micrograms per milliliter alpha-tocopherol protected spermatozoa against lipid peroxidation during incubation, but 1000 microg/mL failed to protect after 2 h of incubation. There was a negative association between TBARS and motility, suggesting that lipid peroxidation affected sperm motility. Both control and 200 microg/mL alpha-tocopherol samples preserved the capacity to generate oxidative energy up to 1 h of incubation. The addition of 200 microg/mL alpha-tocopherol in the semen extender could be useful to preserve boar spermatozoa against the oxidative stress generated by cryopreservation.     

Phospholipase-induced modulation of dolichyl-phosphomannose synthase activity

Rat liver dolichyl-phosphomannose synthase is optimally active when the enzyme is reconstituted with lipids that prefer a nonlamellar macroscopic organization in isolation, such as phosphatidylethanolamine (PE), but the enzyme is only negligibly active in the presence of lipids that normally form stable bilayers, such as phosphatidylcholine (PC) [Jensen, J.W., & Schutzbach, J.S. (1985) Eur. J. Biochem. 153, 41-48]. We now report that the activity of the synthase can be modulated by incorporating diacylglycerol and lysophosphatidylcholine into the lipid matrix. Enzyme activity in PC bilayers was stimulated by the presence of diacylglycerol, a lipid that has a conical dynamic molecular shape and disrupts bilayer stability. In PC/diacylglycerol mixtures the apparent Km for dolichyl-P was 30-fold lower than the apparent Km for the polyprenol acceptor in PC membranes. Enzyme activity was also stimulated when diacylglycerol was generated in situ by incubation of PC vesicles with phospholipase C. In contrast, the activity of enzyme reconstituted in PE dispersions, or in PE/PC bilayers, was markedly inhibited by the presence of lysophospholipids. Enzyme activity was also reduced by the in situ generation of lysophospholipids in PE/PC vesicles by incubation with phospholipase A2. Since lysophospholipids and diacylglycerols arise in vivo as products of phospholipid metabolism, modulation of enzyme activity by these compounds may represent a potential regulatory mechanism for the synthesis of oligosaccharide lipids.     

Phosphatidylcholine metabolism in endothelial cells: evidence for phospholipase A and a novel Ca2+-independent phospholipase C

The metabolism of phosphatidylcholine (PC) was investigated in sonicated suspensions of bovine pulmonary artery endothelial cells and in subcellular fractions using two PC substrates: 1-oleoyl-2-[3H]oleoyl-sn-glycero-3-phosphocholine and 1,2-dipalmitoyl-sn-glycero-3-phospho[14C]choline. When these substrates were incubated with the whole cell sonicate at pH 7.5, all of the metabolized 3H label was recovered in [3H]oleic acid (95%) and [3H]diacylglycerol (5%). All of the 14C label was identified in [14C]lysoPC (92%) and [14C]phosphocholine (8%). These data indicated that PC was metabolized via phospholipase(s) A and phospholipase C. Substantial diacylglycerol lipase activity was identified in the cell sonicate. Production of similar proportions of diacylglycerol and phosphocholine and the low relative activity of phospholipase C compared to phospholipase A indicated that the phospholipase C-diacylglycerol lipase pathway contributed little to fatty acid release from the sn-2 position of PC. Neither phospholipase A nor phospholipase C required Ca2+. The pH profiles and subcellular fractionation experiments indicated the presence of multiple forms of phospholipase A, but phospholipase C activity displayed a single pH optimum at 7.5 and was located exclusively in the particulate fraction. The two enzyme activities demonstrated differential sensitivities to inhibition by p-bromophenacylbromide, phenylmethanesulfonyl fluoride and quinacrine. Each of these agents inhibited phospholipase A, whereas phospholipase C was inhibited only by p-bromophenacylbromide. The unique characteristics observed for phospholipase C activity towards PC indicated the existence of a novel enzyme that may play an important role in lipid metabolism in endothelial cells.     

Spontaneous expression of C-type virus in DBA/2 mice is associated with an increased rate of mortality, independent of neoplastic disease

Ecotropic C-type retroviruses isolated from both normal and dimethylbenzanthracene-treated DBA/2 mice could be classified into three major groups, Ea, Eb, and Ec, that differed in structure and biology. Weanling DBA/2 mice were generally free of viruses, but a fraction of adult individuals became virus positive and were apparently selectively associated with a high expression of the Eb viruses. Some of the ecotropic viruses from DBA/2 mice acted as exogenous pathogens. They caused viremia and a moderate incidence of leukemia when injected into C3H and ST/a mice. In addition, they caused an appreciable number of early deaths without signs of malignancy. To evaluate the natural role of the viruses, we studied the survival of spontaneously viremic and nonviremic DBA/2 mice. The viremic animals as a group were characterized by a significantly reduced life-span that was not related to neoplasia. These observations indicated that endogenous C-type retroviruses can be pathogenic without preselection of the host for disease. They also emphasize that endogenous viruses, like their exogenous counterparts, can have a broader pathogenic spectrum than normally appreciated.     

Mass Spectrometry Reveals New Insights into the Production of Superoxide Anions and 4‐Hydroxynonenal Adducted Proteins in Human Sperm

The free‐radical theory of male infertility suggests that reactive oxygen species produced by the spermatozoa themselves are a leading cause of sperm dysfunction, including loss of sperm motility. However, the field is overshadowed on several fronts, primarily because: i) the probes used to measure reactive oxygen species (ROS) are imprecise; and ii) many reports suggesting that oxygen radicals are detrimental to sperm function add an exogenous source of ROS. Herein, a more reliable approach to measure superoxide anion production by human spermatozoa based on MS analysis is used. Furthermore, the formation of the lipid‐peroxidation product 4‐hydroxynonenal (4‐HNE) during in vitro incubation using proteomics is also investigated. The data demonstrate that neither superoxide anion nor other free radicals that cause 4‐HNE production are related to the loss of sperm motility during incubation. Interestingly, it appears that many of the 4‐HNE adducted proteins, found within spermatozoa, originate from the prostate. A quantitative SWATH analysis demonstrate that these proteins transiently bind to sperm and are then shed during in vitro incubation. These proteomics‐based findings propose a revised understanding of oxidative stress within the male reproductive tract.     

Intracellular transport of phosphatidic acid and phosphatidylcholine into lipid bodies in an oleaginous fungus, Mortierella ramanniana var. angulispora

Exogenous fluorescent phosphatidic acid (PA) and phosphatidylcholine (PC) were transported into lipid bodies in an oleaginous fungus, Mortierella ramanniana var. angulispora [Kamisaka et al. (1999) Biochim. Biophys. Acta 1438, 185-198]. We further investigated the processes of fluorescent PA and PC transport into lipid bodies in this fungus by changing culture conditions. Lowering incubation temperature decreased lipid body labeling by 1-palmitoyl, 2-[5-(5,7-dimethyl boron dipyrromethene difluoride)-1-pentanoyl]-PA (C5-DMB-PA), but fluorescence did not accumulate in organelles other than lipid bodies. C5-DMB-PC transport into lipid bodies was blocked at temperatures below 15 degrees C and fluorescence accumulated in intracellular membranes, presumably endoplasmic reticulum membranes. The low-temperature block of C5-DMB-PC transport enabled us to do pulse-chase experiments in which fungal cells were pulse-labeled at 15 degrees C with C5-DMB-PC and chased at 30 degrees C. The results clearly depicted transport of C5-DMB-PC and its derivatives from intracellular membranes to lipid bodies. Transport was temperature-dependent and ATP-dependent, although microtubules and actin filaments were not substantially involved. Experiments using 14C-labeled fatty acids and glycerol instead of C5-DMB-PC under the same conditions suggested that transport depicted by fluorescence agreed with metabolism and transport of PC containing native fatty acids. Furthermore, the transport mechanism preferred PC containing unsaturated fatty acids such as linoleic acid. This study dissect lipid transport of PA and PC into lipid bodies and reveal regulatory steps for lipid body formation in this fungus.     

Mechanical properties of a biodegradable bone regeneration scaffold

Poly (Propylene Fumarate) (PPF), a novel, bulk erosion, biodegradable polymer, has been shown to have osteoconductive effects in vivo when used as a bone regeneration scaffold (Peter, S. J., Suggs, L. J., Yaszemski, M. J., Engel, P. S., and Mikos, A. J., 1999, J. Biomater. Sci. Polym. Ed., 10, pp. 363-373). The material properties of the polymer allow it to be injected into irregularly shaped voids in vivo and provide mechanical stability as well as function as a bone regeneration scaffold. We fabricated a series of biomaterial composites, comprised of varying quantities of PPF, NaCl and beta-tricalcium phosphate (beta-TCP), into the shape of right circular cylinders and tested the mechanical properties in four-point bending and compression. The mean modulus of elasticity in compression (Ec) was 1204.2 MPa (SD 32.2) and the mean modulus of elasticity in bending (Eb) was 1274.7 MPa (SD 125.7). All of the moduli were on the order of magnitude of trabecular bone. Changing the level of NaCl from 20 to 40 percent, by mass, did not decrease Ec and Eb significantly, but did decrease bending and compressive strength significantly. Increasing the beta-TCP from 0.25 g/g PPF to 0.5 g/g PPF increased all of the measured mechanical properties of PPF/NVP composites. These results indicate that this biodegradable polymer composite is an attractive candidate for use as a replacement scaffold for trabecular bone.     

Lipid peroxidation and phospholipase A2 activity in liposomes composed of unsaturated phospholipids: a structural basis for enzyme activation

The effect of lipid peroxidation on membrane structure and phospholipase A2 activity was studied using liposomes composed of bovine liver phosphatidylcholine (PC) and phosphatidylethanolamine (PE). The phospholipids were mixed at set ratios and sonicated to yield small unilamellar vesicles. The liposome preparations were subjected to lipid peroxidation as induced by cumene hydroperoxide and hematin. Under these conditions, a sharp increase in lipid peroxidation was noted over a 30 min incubation period and was accompanied by loss of polyunsaturated fatty acids (PUFA). Liposomes enriched in PE were most extensively peroxidized with a preferred oxidation of this phospholipid. The extent of PC oxidation was also greater in liposomes containing the largest proportions of PE. Analysis of liposome anisotropy, via steady-state fluorescence polarization of diphenylhexatriene indicated that progressive increases in either PE content or the level of lipid peroxidation increased the apparent microviscosity of the vesicles. Moreover, lipid peroxidation increased anisotropy more effectively than variations in the ratios of PE vs. PC. Thus, peroxidation of 5-10% of the phospholipids produced the same anisotropy increase as a 20% increase in the ratio of PE vs. PC. Analysis of vesicle turbidity suggested that fusion was also more readily achieved through lipid peroxidation. When liposomes were incubated with 0.4 U/ml of snake venom phospholipase A2, a direct correlation was found between the degree of lipid peroxidation and the extent of phospholipid hydrolysis. The more unsaturated phospholipid, PE, was most extensively hydrolyzed following peroxidation. Increasing the proportion of PE also resulted in more extensive phospholipid hydrolysis. These findings indicate that lipid peroxidation produces a general increase in membrane viscosity which is associated with vesicle instability and enhanced phospholipase A2 attack. A structural basis for membrane phospholipase A2 activation as a consequence of lipid peroxidation is discussed in light of these findings.     

Role of seminal plasma in damage to turkey spermatozoa during in vitro storage

In vitro storage of turkey spermatozoa is performed without consideration of the potential role of seminal plasma on sperm functions. We report the effects of seminal plasma on membrane permeability, lipid metabolism, energy status, motility and fertility of turkey spermatozoa stored at 4 or 20 degrees C. Phospholipid content (1077 nmol/10(9) spz versus 1219 nmol/10(9) spz at 48 h) and membrane permeability of spermatozoa were significantly damaged by the presence of seminal plasma after 48 h of storage at 4 degrees C, whereas damage to ATP content and fertility occurred earlier damaged by this presence (fertility after 24h storage 51% with seminal plasma versus 71% without). At 20 degrees C, seminal plasma decreased the phospholipid content of spermatozoa in the first hour of storage (1326 nmol/10(9) spz versus 1636 nmol/10(9) spz). Twenty-four hours later, this effect was masked by intense lipid peroxidation. These results show that seminal plasma is deleterious to storage of turkey spermatozoa at 4 degrees C and is involved in phospholipid metabolism of spermatozoa. Lipid peroxidation could be responsible for the acceleration of the degradation of sperm phospholipids during storage at 20 degrees C. However, lipid peroxidation seems not to be active at 4 degrees C. In this case, we suggest that phospholipase activation may contribute to sperm degradation, especially in the presence of seminal plasma.     

Endogenous substrate for energy metabolism and ultrastructural correlates in spermatozoa of the sea urchin Diadema setosum

Energy metabolism in spermatozoa of the sea urchin Diadema setosum of the order Diadematoida was examined. The spermatozoa contained not only several kinds of phospholipids and cholesterol, but also triglyceride (TG). Glycogen and glucose were present at extremely low levels. Following dilution of dry sperm and incubation in seawater, the TG content decreased rapidly. Other lipids, however, remained at constant levels, except for a slight increase in the level of free fatty acid. High lipase activity was demonstrated in the spermatozoa. ¹⁴C-labeled fatty acid was oxidized to ¹⁴CO₂. Ultrastructural study also showed that lipid globules were present at the bottom of the midpiece. After incubation in seawater, morphological changes in the lipid globules were observed and some vacuoles appeared. Thus, the results obtained strongly suggest that D. setosum spermatozoa obtain energy through oxidation of fatty acid from TG stored in the lipid globules at the midpieces. © 1995 Wiley-Liss, Inc.