Microtubules in relation to the motility of a sperm syncytium in an armored scale insect.

Male scale insects of the species Parlatoria oleae Colvée (Homoptera: Coccoidea) produce motile sperm bundles. The bundle is a syncytium consisting of 10 to 20 closely packed, filamentous spermatozoa, which share a common cytoplasm and are enclosed in a common membrane. The individual spermatozoon is not surrounded by a plasma membrane, but is delimited by a scroll-like sheath composed of 45 to 50 microtubules. The microtubules run parallel to the long axis of the spermatozoon and are arranged in a spiral pattern as seen in transection. The outside diameter measures approximately 140 to 220 A and the inside diameter, 70 to 100 A. The spermatozoon is about 300 micro long and tapers gradually from a diameter of approximately 0.3 micro anteriorly to 0.1 micro posteriorly. The anterior half (150 micro) has a threadlike core of chromatin about 0.07 micro in diameter. A homogeneous cytoplasm surrounds the nuclear core and fills the posterior half of the spermatozoon. Neither osmium tetroxide nor glutaraldehyde fixation revealed the presence of a nuclear envelope, acrosomal membranes, mitochondria, flagellum, or centrioles. In spite of the apparent lack of orthodox cell organelles, the spermatozoon is actively motile upon release from the bundle. It exhibits capactiy for motility throughout its entire length. Since the sheath of microtubules is the only structure which extends the full length of the spermatozoon, it probably plays a significant role in spermatozoan motility.

Microtubular patterns in spermatozoa of coccid insects in relation to bending.

Flagella-like motion occurs in filamentous spermatozoa of coccid insects, which have diameters (0.16-0.65 micro) and lengths (150-300 micro) similar to those of long flagella, but have no doublets or 9 + 2-like arrangements of microtubules. Light and electron microscope investigations of spermatozoa from 10 species reveal many bizarre patterns of microtubules and suggest some basic similarities to flagella. Detailed analyses of spermatozoa which are naturally bent in definable planes during their elongation in the male and their storage in the female provide evidence that a constant topographical relationship is maintained between their unorthodox patterns of microtubules, as viewed in transections, and the direction of bending. The configuration common to most coccid spermatozoa consists of an acentrically positioned crescent of microtubules surrounded by one to several concentric rings. A line drawn to connect the two ends of the crescent appears to remain perpendicular to the plane of bending, and it defines a plane in which bisection of the spermatozoon produces halves with unequal numbers of microtubules. Bisection of the 9 + 2 motile apparatus in a plane perpendicular to that of bending also appears to produce halves with unequal numbers of microtubules. Therefore, the indispensable elements for flagellar and flagella-like motion may be microtubules arranged in "asymmetric" patterns.

Unusual microtubular patterns and three-dimensional movement of mealybug sperm and sperm bundles.

The spermatozoon of the mealybug Pseudococcus obscurus Essig is a filamentous cell (0.25 micro by 300 micro) which exhibits three-dimensional flagellations throughout most of its length. It has microtubules (200 A diameter) and a threadlike nuclear core (0.07-0.09 micro diameter) which extend almost its entire length, but apparently it has no mitochondria, centrioles, typical flagellum, or acrosome. The microtubules are arranged in two and a half concentric rings and total 56 in the most actively motile region but form two or three concentric rings with totals of 28 or 56 tubules, respectively, in less active regions. The relation of unusual microtubular patterns to the 9 + 2 complex and to flagellar motion is discussed. Mealybug spermatozoa are transmitted to the female in motile bundles which are approximately 1.3 micro by 750 micro and have four regions: (1) an anterior corkscrew region; (2) a region which contains approximately 16 spermatozoa; (3) a region of amorphous content; and (4) an endpiece. Bundle motility originates from the synchronous movements of its spermatozoa which appear to be arranged in two concentric multistranded helices. The spermatozoa provide both forward and gyratory motions of the bundle, and the corkscrew complements bundle propulsion by converting part of the rotation into forward movement.

Effects of cyclic AMP and Sephadex fractions of chick embryo extract on cloned retinal pigmented epithelium in tissue culture.

The effects of dibutyryl cyclic 3',5'-adenosine monophosphate (BcAMP) and Sephadex G-25 fractions of chick embryo extract on the growth rate, morphology, and pigmentation of normal chick retinal pigmented epithelium (PE) were investigated. Seven cloned PE cell lines were each grown in modified Ham's F-12 medium alone (F-12), or in F-12 supplemented with either high molecular weight (H) or low molecular weight (L) fractions of chick embryo extract. Cells grown in F-12 alone or in L medium formed compact epithelial sheets, whereas cells grown in H had a fibrocytic appearance and formed poorly organized monolayers. In H plus BcAMP, cell morphology was more epithelioid than in H alone, and generally the monolayers appeared more differentiated. Under each of these three culture conditions, 2 x 10(-4) M BCAMP retarded the increase in cell number and decreased the final number of cells per culture dish, but had little effect on plating efficiency. BcAMP also increased the rate of cell adhesion to a plastic substratum. Pigmentation was marked in cultures grown in F-12 or in L alone, but the addition of BcAMP dramatically reduced visible pigmentation. This effect was reversed when BcAMP was removed from the culture medium. Thus BcAMP modifies cell and colonial morphology, rate of cell accumulation, adhesive properties, and pigmentation of normal PE cells.

Retinal capillaries: proliferation of mural cells in vitro.

Capillaries from bovine, monkey, and human retinas maintained in tissue culture produced a monolayer of cells. Autoradiographic and electron microscopic evidence indicated that the mural cells (intramural pericytes) were the cells that proliferated. Since intramural pericytes are damaged selectively in diabetes mellitus, their availability in culture will be useful in seeking means to control diabetic retinopathy.

Retinal capillaries: basement membrane thickening by galactosemia prevented with aldose reductase inhibitor.

A twofold thickening of capillary basement membranes of rat retinas resulting from dietary galactose was prevented by sorbinil, an inhibitor of aldose reductase. Since the basement membrane thickening was ultrastructurally similar to that typical of diabetic retinopathy, it may indicate changes in vessel permeability and susceptibility to hemorrhage. Galactosemic rats should be useful models for studying basement membrane-related complications of diabetes and for examining the potential biochemical regulation of basement membrane synthesis by aldose reductase inhibitors.

Dietary vitamins A and E influence retinyl ester composition and content of the retinal pigment epithelium.

Experiments were conducted to determine the influence of dietary levels of vitamin A and alpha-tocopherol on the amounts and composition of retinyl esters in the retinal pigment epithelium of light-adapted albino rats. Groups of rats were fed diets containing alpha-tocopherol and either no retinyl palmitate, adequate retinyl palmitate, or excessive retinyl palmitate. Other groups of rats received diets lacking alpha-tocopherol and containing the same three levels of retinyl palmitate. Retinoic acid was added to diets lacking retinyl palmitate. After 27 weeks, the animals were light-adapted to achieve essentially total visual pigment bleaches, and the neural retinas and retinal pigment epithelium-eyecups were then dissected from each eye for vitamin A ester determinations. Almost all of the retinyl esters were found in the retinal pigment epithelium-eyecup portions of the eyes, mainly as retinyl palmitate and retinyl stearate. Maintaining rats on a vitamin A-deficient, retinoic acid-containing diet led to significant reductions in retinal pigment epithelial retinyl ester levels in rats fed both the vitamin E-supplemented and vitamin E-deficient diets; contrary to expectations, the effect of dietary vitamin A deficiency was more pronounced in the vitamin E-supplemented rats. Vitamin A deficiency in retinoic acid-maintained animals also led to significant reductions in retinyl palmitate-to-stearate ester ratios in the retinal pigment epithelia of both vitamin E-supplemented and vitamin E-deficient rats. Excessive dietary intake of vitamin A had little, if any, effect on retinal pigment epithelial retinyl ester content or composition. Vitamin E deficiency resulted in significant increases in retinal pigment epithelial retinyl palmitate content and in palmitate-to-stearate ester ratios in rats fed all three levels of vitamin A, but had little effect on retinal pigment epithelial retinyl stearate content. In other tissues, vitamin E deficiency has been shown to lower vitamin A levels, and it is widely accepted that this effect is due to autoxidative destruction of vitamin A. The increase in retinal pigment epithelial vitamin A ester levels in response to vitamin E deficiency indicates that vitamin E does not regulate vitamin A levels in this tissue primarily by acting as an antioxidant, but rather may act as an inhibitor of vitamin A uptake and/or storage. The effect of vitamin E on pigment epithelial vitamin A levels may be mediated by the vitamin E-induced change in retinyl palmitate-to-stearate ratios.

Prevention of basement membrane thickening in retinal capillaries by a novel inhibitor of aldose reductase, tolrestat.

A diabetic-like thickening of retinal capillary basement membranes induced in rats fed for 207 consecutive days a diet containing 50% galactose was prevented by the addition to the diet of tolrestat, a potent, structurally novel inhibitor of aldose reductase. Analysis of electron micrographs (X 25,000) of capillaries from the outer plexiform layer of the retina by computer planimetry showed that the basement membranes were approximately twofold thicker in rats fed galactose than in those fed either a standard diet or a diet containing galactose and tolrestat in doses of 43 or 57 mg/kg/day. The thickening of basement membranes in galactose-fed rats was accompanied by other ultrastructural alterations mimicking changes typical of diabetic microangiopathy, such as multilamination and the formation of vacuoles and dense inclusions. Therefore, the galactosemic rat represents a useful model for studying basement membrane-related complications of diabetes and their possible prevention by aldose reductase inhibitors.

Senescence and the retinal pigment epithelium: alterations in basal plasma membrane morphology.

Marked age-related changes in the morphology of retinal pigment epithelial (RPE) cell basal infoldings were found in pigmented rats. Quantitative morphometric analysis revealed that during senescence the amount of basal plasma membrane per unit RPE length increased substantially, the regional distribution of the basal infoldings along the RPE became more irregular, and the average depth of penetration of the basal infoldings into the RPE increased dramatically. The crucial role of the RPE in maintaining retinal integrity suggests that the observed changes in RPE morphology may be involved in the development of senile retinopathies which occur in a variety of species, including man.

Relationship between dietary retinol and lipofuscin in the retinal pigment epithelium.

A variety of evidence suggests that autoxidation of cellular components probably plays a significant role in the age-related accumulation of lipofuscin, or age-pigment, in the mammalian retinal pigment epithelium (RPE). Among the likely candidates for conversion into RPE lipofuscin fluorophores via autoxidative mechanisms are vitamin A compounds, which are present in the retina and RPE in high concentrations. Vitamin E, an important lipid antioxidant, is likely to inhibit vitamin A autoxidation. Experiments were conducted to evaluate the significance of vitamin A autoxidation in the deposition of lipofuscin in the RPE. Albino rats were fed diets either supplemented with or lacking vitamin E. Each of these two groups of animals was further subdivided into three groups which were fed different levels of vitamin A palmitate: none, 14.0 mumol/kg diet, and 80.5 mumol/kg diet. After 26 weeks, the animals were killed and the RPE lipofuscin contents were determined by both fluorescence measurements and quantitative ultrastructural morphometry. Vitamin A palmitate deficiency led to significant reductions in RPE lipofuscin deposition, relative to the amounts of this pigment present in the groups receiving vitamin A palmitate in their diets. The relative magnitude of the vitamin A effect was greater in the vitamin E-supplemented groups than in the groups fed the diets deficient in vitamin E. This finding suggests that vitamin E interacts with vitamin A ester metabolites in vivo in a more complex manner than simply acting as an antioxidant protectant. Rats fed the diets containing the higher level of vitamin A palmitate failed to display elevated RPE lipofuscin contents relative to those in the rats fed 14.0 mumol of vitamin A palmitate/kg diet. Failure of high vitamin A intake to enhance RPE lipofuscin deposition may have been due to the fact that intake of vitamin A above normal levels did not lead to an elevation in vitamin A content of the retinal tissue. Establishing an effect of vitamin A deficiency on RPE lipofuscin deposition and characterization of the interactions between vitamins E and A are important steps toward defining precisely the molecular and cellular mechanisms underlying age-pigment accumulation in the RPE.

Lipofuscin autofluorescence: evidence for vitamin A involvement in the retina.

A lipofuscin-like autofluorescence develops in the degenerating photoreceptor cells of the RCS rat, a strain with inherited retinal dystrophy. In animals with normal retinas, age-related lipofuscin accumulation in the eye is restricted to the retinal pigment epithelium (RPE). Previous investigations have established that RPE lipofuscin accumulation in the normal rat retina can be reduced by dietary vitamin A deficiency. In order to determine whether the photoreceptor-derived fluorescence in the RCS rat retina is related to RPE lipofuscin fluorescence, the influence of dietary vitamin A on the fluorophore content of the RCS rat retina was studied. Vitamin A deficiency substantially reduced the autofluorescence associated with degenerating photoreceptor cells of the RCS rat retina. A specific vitamin A-dependent fluorophore was isolated from these retinas using thin-layer chromatography (TLC). The mobility of this fluorophore on TLC differs from that of the major age-dependent fluorophore isolated from the RPE of normal rats. Thus, if the vitamin A-dependent fluorophores of the photoreceptors and RPE are related, it appears that the fluorophore generated in the photoreceptor cells must undergo chemical modification once it has been taken up by the RPE. The fact that both the RPE- and photoreceptor-associated fluorophores are vitamin A-dependent suggests that such a relationship between them is likely. These experiments indicate that the RPE is somewhat different from other lipofuscin-accumulating tissues in that a major precursor of RPE lipofuscin fluorophores originates in another cell type and enters the RPE via phagocytosis.

Lipofuscin accumulation resulting from senescence and vitamin E deficiency: spectral properties and tissue distribution.

The corrected fluorescence emission spectra and tissue distributions of the autofluorescent pigments which accumulate during normal aging and as a consequence of vitamin E deficiency were studied in albino rats. In the retinal pigment epithelium, both the age-related pigment (lipofuscin) and the pigment related to vitamin E deficiency had essentially identical emission spectra. Peak emission occurred from 590 to 650 nm. Young animals which had been kept on a vitamin E deficient diet for 17 weeks after weaning showed significant accumulations of autofluorescent pigment in uterus, duodenum, and retinal pigment epithelium, but not in the spinal cord or inferior olivary nucleus of the brain. Old animals (96 weeks) fed a commercial diet with adequate vitamin E had accumulated lipofuscin in the retinal pigment epithelium, spinal cord gray matter, and inferior olivary nucleus, but not in the duodenum or uterus. Thus, while the auto-fluorescent pigments related to aging and vitamin E deficiency have similar properties, their tissue distributions are quite different.

A new, albino-beige mouse: giant granules in retinal pigment epithelium.

Albino-beige mice were produced in order to combine two experimentally useful characteristics, albinism and lysosomal dysfunction, in the same animal. The retinal pigment epithelium of albino-beige mice formed giant intracellular granules. Exposure of albino-beige mice to white light of 150 foot-candles for 3 to 10 hr induced marked phagocytosis of rod outer segment fragments by the retinal pigment epithelium, resulting in intracellular accumulations of undigested disk membranes within the giant granules. Additional, incompletely processed membranes accumulated as the mice aged or were exposed to 150 foot-candle light for longer periods. Such accumulations of ingested membranes were not observed in the pigment epithelium of exposed or aging albino mice heterozygous for the beige gene. Because of its altered processing of ingested outer segment membranes, this new albino mouse should be useful for studying the possible roles of the retinal pigment epithelium in the maintenance of photoreceptor cells and in their recovery from light damage and other insults.

Vitamin A storage and peroxisomes in retinal pigment epithelium and liver.

Retinas and livers were studied with histochemical methods for catalase combined with light and electron microscopy, following intramuscular injections of C57 black mice with retinyl acetate or retinyl palmitate in total doses of 0.9 to 6.7 X 10(6) I.U./kg. body weight. There were clear, dose-related increases in the numbers and sizes of vitamin A--storing lipid droplets in the stellate cells of the liver. Concomitantly, more conservative increases in similar lipid droplets occurred in the pigment epithelium but not in other cells of the retina. Such lipid droplets may represent physiological sites of vitamin A storage which are important for the maintenance of photoreceptor cells by the retinal pigment epithelium. No changes in lipids of the retina or liver were observed in mice injected with retinoic acid or with peanut oil. Both peroxisomes containing catalase and the putative vitamin A--storing lipid droplets were distributed along the basal and lateral cell surfaces of the pigment epithelium where receptors for plasma retinol-binding protein have been reported. Peroxisomes may play a role in the reactions related to the esterification and sequestering of vitamin A.

Aldose reductase, diabetes, and thickening of the retinal inner limiting membrane.

Capillary basement membrane thickening is typical of the diabetic retina, and aldose reductase appears to be involved since a diabetic-like thickening can be induced by galactose feeding and prevented with aldose reductase inhibitors. Because aldose reductase is present in the Mueller's cells, studies were undertaken to determine if thickening of the retinal inner limiting membrane, which is the basement membrane of these cells, can be induced by long-term galactose feeding and be prevented with an aldose reductase inhibitor. Weanling male, Sprague-Dawley rats were given a 50% galactose diet with or without an aldose reductase inhibitor (0.04% tolrestat, Ayerst). Quantitative computer planimetry on electron micrographs demonstrated a significant galactose-induced thickening of the inner limiting membrane which was prevented by the aldose reductase inhibitor. The results were consistent with the notion that basement membrane thickening is involved in diabetic retinopathy and can be delayed or prevented with aldose reductase inhibitors.

Deficiencies of vitamins E and A in the rat. Retinal damage and lipofuscin accumulation.

The interrelationships of vitamins E and A in maintaining various structural components of the retina were studied in four groups of weanling female rats fed purified diets adequate or deficient in each vitamin: +E, +A; -E, +A; +E, -A; and -E, -A. Groups deficient in retinol (-A) were supplemented with retinoic acid. After 14, 21, and 35 weeks, the retinas were examined histologically and ultrastructurally. At 35 weeks, the doubly deficient rats (-E, -A) had lost 92% of their rod nuclei, whereas rats deficient in vitamins A (+E, -A) or E (-E, +A) alone had lost only 34% and 20%, respectively. Vitamin E deficiency resulted in extensive lipofuscin deposits in the retinal pigment epithelium as early as 21 weeks, but the presence of vitamin A doubled the number of lipofuscin granules (-E, +A vs. -E, -A) and induced an even greater increase in their autofluorescence. Another clear influence of vitamin A was seen when +E, +A retinas autofluoresced not only much more than +E, -A retinas, which had similar numbers of granules, but also more than -E, -A retinas, which had about twice as many lipofuscin granules. In the retina, unlike the uterus, the lipofuscin-specific autofluorescence and lipofuscin granule number were not proportional. Moreover, the numbers of granules were influenced by both vitamins E and A, whereas the intensity of lipofuscin-specific autofluorescence was determined almost exclusively by vitamin A. Probably the accelerated loss of photoreceptor cells in -E, -A retinas resulted from both oxidation of membranes and oxidation of retinal vitamin A stores in the absence of vitamin E protection.

Vitamin E deficiency and the retina: photoreceptor and pigment epithelial changes.

To investigate the role of normal vitamin E levels and the interrelationships between vitamin E and A in maintaining the visual cells of the retina, weanling female Sprague-Dawley rats were fed vitamin E-free diets differing tenfold in their vitamin A content (0.8 and 8.0 mg of retinol per kilogram of diet). Rats on vitamin E-free diets with the higher vitamin A level exhibited marked disruption of photoreceptor outer segment membranes and a fivefold increase in the number of lipofuscin granules in the pigment epithelial cells which ingest these membranes. Rats on vitamin E-free diets with the lower vitamin A level showed the same retinal damages plus significant loss of photoreceptor cells compared to age-matched rats on control diets. Rods and cones were involved equally, and their pattern of loss was not like that found in vitamin A deficiency. Normal levels of vitamin E probably protect photoreceptor membranes from oxidative damage and retard the accumulation of their remnants and other products of lipid breakdown in the pigment epithelium. The vitamin A status of rats has a significant influence on the extent of damage induced by vitamin E deficiency.

Age-related thickening of retinal capillary basement membranes.

Capillary basement membrane thickness is known to increase in a variety of tissues during aging and as a consequence of certain systemic disease states. In order to determine whether capillary basement membrane thickness increases during senescence in the outer plexiform (OPL) and ganglion cell (GCL) layers of the retina, eyes of pigmented ACI rats aged 4, 11, 17, and 32 mo were examined. Basement membrane thickness in the OPL and GCL were determined at each of these ages using computer-assisted ultrastructural morphometry. Capillary basement membranes in both the OPL and GCL showed a linear increase in thickness over the 4 to 32 mo age span. The increase in thickness averaged 54% for the OPL and 65% for the GCL over this age range. At all ages examined, the GCL basement membranes were 80 to 90% thicker than those in the OPL. Age-related changes in the substructure of the capillary basement membranes differed between the OPL and GCL. It is possible that the observed age-related changes in retinal capillary basement membranes may play a role in the development of senile retinopathies.

Novel procedures for isolating intact retinal vascular beds from diabetic humans and animal models.

PURPOSE: To improve the 30-year-old "trypsin digestion" procedure for isolation of the complete retinal vasculature, which, in its time, was a revolutionary advance that allowed important discoveries about diabetic retinopathy; to provide a method that will yield more consistent results when applied to retinas representing a wide range of ages, species, and severity of vascular disease, such as that occurring in diabetes. METHODS: Because the Difco trypsin preparation (Difco Laboratories, Detroit, MI) is a crude pancreatic extract, containing variable amounts of chymotrypsin, elastase, amylase, lipase, ribonuclease, collagenase, and other contaminants, an attempt was made to determine which of the major enzymes alone (using purified preparations), or what combination of enzymes, might be most effective in providing consistently clean yet intact retinal vasculatures from eyes of different origins. RESULTS: Purified elastase alone (40 U/ml) in 100 mmol/l sodium phosphate buffer with 150 mmol/l sodium chloride and 5 mmol/l EDTA at pH 6.5 and 37 degrees C gave better results than various concentrations of purified trypsin or chymotrypsin alone, or mixtures of trypsin/chymotrypsin, trypsin/elastase, chymotrypsin/elastase, or the crude trypsin preparation. CONCLUSIONS: Elastase, which exhibits broad protease activity, and not trypsin, is the most important enzyme of the standard, crude trypsin digestion procedure for removal of the nonvascular tissues of the retina.

Deficiencies of vitamins E and A in the rat: lipofuscin accumulation in the choroid.

The effects of vitamin E and A deficiencies on the formation of lipofuscin in the melanocytes and fibroblasts of the choroidal stroma and in the endothelial cells of the choriocapillaris were studied. Weanling female albino rats (Sprague-Dawley) were divided into three groups and fed purified diets adequate or deficient in vitamins E and A: +E, +A; -E, -A. After 35 weeks, vitamin E deficient rats (-E, +A) exhibited increased lipofuscin-specific autofluorescence in the choroid compared to the controls (+E, +A). By electron microscopy and morphometric methods the choroidal stroma of vitamin E deficient rats displayed an increase in lipofuscin content as measured by the number of lipofuscin granules and their size, if compared with the controls. However, in vitamin E deficiency, only animals with a supply of vitamin A (-E, +A) showed higher amounts of lipofuscin in the choroidal stroma; animals deficient in both vitamins (-E, -A) stayed on the same level as the controls (+E, +A). In the endothelial cells of the choriocapillaris, on the other hand, no significant increases in lipofuscin content were observed in either group of vitamin E deficient animals (-E, +A), (-E, -A). Apparently vitamin E deficiency affects the choroid by increasing lipofuscin formation only in the melanocytes and fibroblasts. Vitamin A appears to play a role in lipofuscin formation.