July 1999
Volume 40, Issue 8
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Retinal Cell Biology  |   July 1999
Marked Alteration of Sterol Metabolism and Composition without Compromising Retinal Development or Function
Author Affiliations
  • Steven J. Fliesler
    From the Department of Ophthalmology, Saint Louis University Eye Institute; the
    Program in Cell and Molecular Biology, Saint Louis University School of Medicine, Missouri; the
  • Michael J. Richards
    From the Department of Ophthalmology, Saint Louis University Eye Institute; the
  • Chi-yen Miller
    From the Department of Ophthalmology, Saint Louis University Eye Institute; the
  • Neal S. Peachey
    Research Service, Hines Veterans Administration Hospital, Illinois; and the Departments of
    Neurology and
    Ophthalmology, Stritch School of Medicine, Loyola University of Chicago, Maywood, Illinois.
Investigative Ophthalmology & Visual Science July 1999, Vol.40, 1792-1801. doi:
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      Steven J. Fliesler, Michael J. Richards, Chi-yen Miller, Neal S. Peachey; Marked Alteration of Sterol Metabolism and Composition without Compromising Retinal Development or Function. Invest. Ophthalmol. Vis. Sci. 1999;40(8):1792-1801.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

purpose. To evaluate the consequences of altering retinal sterol metabolism and composition on the development, histologic organization, and electrophysiological function of the retina, under conditions that mimic the biochemical hallmarks of the Smith–Lemli–Opitz (SLO) syndrome.

methods. Pregnant Sprague–Dawley rats were fed cholesterol-free chow containing AY9944 (treated group), an inhibitor of 3β-hydroxysterolΔ 7-reductase, from gestational day 6 through postnatal day (P)28. Control animals were fed the same chow, but without AY9944. In addition, progeny in the treated group were injected subcutaneously every other day from birth to P28 with an olive oil emulsion containing AY9944; control animals received olive oil emulsion alone. At various postnatal times, tissues from treated and control animals were harvested, and their sterol profiles were analyzed by reversed-phase high-performance liquid chromatography. Companion eyes from animals of both groups were examined histologically at P1. At P28, animals were evaluated by electroretinography; tissues were then harvested for biochemical analysis and companion eyes were subjected to histologic and ultrastructural analyses.

results. Treatment of developing rats with AY9944 caused markedly abnormal accumulation of 7-dehydrosterols and severely reduced cholesterol levels in all tissues examined, relative to control animals. Despite this, treated animals exhibited normal retinal development and had no overt ocular defects or decrease in electroretinographic function, up to P28.

conclusions. These results were unexpected, given the known biophysical effects of such sterol alterations on membrane properties and the profound dysmorphic and cognitive abnormalities associated with genetic defects in 3β-hydroxysterol Δ7-reductase that have been linked to the SLO syndrome. The results suggest that 7-dehydrosterols can substitute functionally for cholesterol in the retina or perhaps can act synergistically with subthreshold levels of residual cholesterol to allow normal cellular structure and function to be achieved.

Cholesterol is the dominant sterol of vertebrate cells and, with rare exception, is required for their structural integrity and viability. 1 2 3 Although sterols other than cholesterol can be incorporated into artificial and cellular membranes, they normally represent minor constituents in mammalian cells and usually cannot support membrane function and cholesterol. 4 5 6 In addition to the more classic roles of cholesterol as a membrane constituent and precursor of bile acids and steroid hormones, members of the hedgehog family of proteins, which are essential signal transduction molecules involved in embryonic development, 7 require covalent sterol lipidation to undergo autocatalytic cleavage and to exert their biologic functions in cells. 8 9  
We have been interested in elucidating the biologic role of cholesterol and other isoprenoids in the development and maintenance of the retina, particularly regarding the morphogenesis and renewal of rod outer segment (ROS) membranes. 10 In the present study, we altered the normal sterol composition of the retina in developing rats, using a metabolic inhibitor (AY9944) that is known to block the cholesterol biosynthetic pathway, so that a precursor (7-dehydrocholesterol), rather than cholesterol, would form and accumulate in the retina. AY9944 is a potent inhibitor of 3β-hydroxysterol Δ7-reductase 11 12 and is known to cause profound teratogenic effects during embryogenesis. 13 14 Therefore, we expected that exposure of animals to this agent from embryonic through early neonatal development would produce dramatic effects on ocular development and function. Contrary to this expectation, however, we report herein that, although AY9944 treatment produced the expected effects on tissue sterol composition, retinal histogenesis and maturation were remarkably normal, and the retina remained electrophysiologically competent under these conditions. 
Materials and Methods
AY9944 Treatment
AY9944 (trans-1,4-bis (2-dichlorobenzylamino-ethyl) cyclohexane dihydrochloride; Wyeth–Ayerst, Wheaton, IL) was mixed with cholesterol-free, powdered rat chow (Ralston Purina, St. Louis, MO), 1 mg AY9944 per 100 g chow. Pregnant Sprague–Dawley rats were fed 40 g chow per day ad libitum throughout the time course of the experiment (gestational day 6 through postnatal day [P]28); control dams received the same chow, minus AY9944. In addition, starting at P1, surviving pups were injected subcutaneously every other day with AY9944 (20 mg/kg) in an olive oil emulsion supplemented with a mixture of vitamins A, D3, and E. To make the olive oil emulsion, one volume of a concentrated AY9944 solution (50 mg/ml, in distilled water) was mixed with four volumes of U. S. Pharmacopeia olive oil, using a dual-syringe microemulsifying device. The olive oil was supplemented with vitamins A, D3, and E by diluting a commercial vitamin preparation (Vital-E-A+D, Schering–Plough Animal Health, Kenilworth, NJ; 1:100 vol/vol) in olive oil (final concentrations, in international units per milliliter: A, 1000; D3, 100; E, 3). 
In our experience, failure to include the vitamin mixture in the vehicle resulted in approximately 75% mortality within the first postnatal week, with few (if any) animals surviving to postnatal week 4 in a given litter. Use of aqueous AY9944 solutions resulted in focal skin lesions and nodules at the injection site, in addition to high mortality. Control pups received vitamin-supplemented olive oil vehicle alone. All animal procedures were approved by the local institutional Animal Care Committees, and were in accordance with the ARVO Resolution on the Use of Animals in Research and with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. 
Lipid Analysis
At various postnatal days, tissues (e.g., serum, liver, retina, brain) from treated and control pups (two to four animals per time point; three experiments) were harvested and saponified, and the nonsaponifiable lipids were extracted and analyzed by reversed-phase high-performance liquid chromatography (HPLC) to quantify the types and amounts of sterols. Methods used were essentially those described previously, 15 except for the commercial system used: reversed-phase column, 150 × 4.6 mm (model IB-SIL 3 C18 BDS; Phenomenex, Torrance, CA); guard column (mobile phase, MeOH at 1 ml/min; detection at 205 nm; NovaPak C18; Waters/Millipore, Milford, MA). Using this system, retention times (relative to those of cholesterol (Δ5, 1.00 minute) for the following authentic isoprenoid lipid reference standards were obtained: cholesta-5,7,24-trien-3β-ol (Δ5,7,24), 0.70 minutes; cholesta-5,24-dien-3β-ol (desmosterol, Δ5,24), 0.79 minutes; cholesta-5,8-dien-3β-ol (8-dehydrocholesterol,Δ 5,8), 0.83 minutes; cholesta-5,7-dien-3β-ol (7-dehydrocholesterol, Δ5,7), 0.89 minutes; squalene, 1.21 minutes. HPLC peak assignments were made in comparison with these reference standards, and each sterol was quantified by integrated peak area analysis in comparison with the empirically determined response factor (integration units per nanomole) for the given standard compound. The relative response factors (relative to cholesterol, 1.00) were as follows: cholesta-5,7,24-trien-3β-ol, 2.78; desmosterol, 2.53; 7-dehydrocholesterol, 1.10; squalene, 12.89. 
Histology
In parallel with the HPLC measurements, contralateral eyes from treated and control animals were fixed, processed for embedding in paraffin or epoxy resin, and examined by light and electron microscopy as described elsewhere in detail. 16 17  
Electroretinography
After overnight dark adaptation, P28 rats were anesthetized (ketamine, 20 mg/kg; xylazine, 2.5 mg/kg; intraperitoneally) and placed on a heating pad. Electroretinograms (ERGs) were recorded as described in detail elsewhere. 18 The amplitude of the a-wave was measured from the prestimulus baseline to the trough of the a-wave. The amplitude of the b-wave was measured to the positive peak, either from the trough of the a-wave or (if no a-wave was present) from the baseline. Implicit times were measured from the time of stimulus presentation to the a-wave trough or the b-wave peak. 
Results
Effect of AY9944 on Neonatal Ocular Histology
Figure 1 shows the cross-sectional gross anatomy of eyes from a P1 control (Fig. 1A) and a P1 AY9944-treated rat (Fig. 1B) . At the gross level, all ocular tissues of the treated eyes appeared comparable to the corresponding tissues in the control eyes. However, treated pups had slightly smaller than normal eye size, consistent with the reduced body mass of the treated animals (73% of control; 4.56 ± 0.32 g; n = 44 from four litters), relative to control animals (6.80 ± 0.40 g; n = 25 from two litters). Other than low birth weight, we did not observe any obvious phenotypic differences between treated and control pups. Histologic examination of retinas at higher magnification (Figs. 1C 1D , respectively) revealed that the cytologic organization of the neural retina in both the treated and control animals was similar and was consistent with the expected histology of a P1 rat retina. 19 20 Subtle differences in the cytologic appearance of the neuroblastic layer suggest that the treated retina may lag slightly in development (i.e., equivalent to a day or less), relative to the control retina. This minor difference aside, there was no evidence for retinal dysplasia, increased pyknosis, or other overt signs of cytologic defects as a consequence of exposure to AY9944. 
Effect of AY9944 on Neonatal Tissue Sterol Composition
Reversed-phase HPLC chromatograms obtained from retinas, brains, and livers of control- and AY9944-treated rats (n = 1 per group) at P1 are shown in Figure 2 . Quantitative data for tissue sterol composition from all animals at P1 and P28 are summarized in Table 1 . Striking differences were observed in the tissue sterol profiles of the treated rats compared with control animals. At P1, cholesterol (Δ5) accounted for approximately 95 mol % of the total sterols in control retinas (Fig. 2A) , with desmosterol (Δ5,24) largely accounting for the remainder; no 7-dehydrosterols were detected. 
In comparison, nearly half of the total sterols in treated retinas (Fig. 2B) were 7-dehydrosterols, whereas cholesterol accounted for approximately 46 mol % of the sterols. (In the given example [Fig. 2B ], the small, unresolved shoulder on the leading edge of theΔ 5,7 peak is consistent with Δ5,24, representing approximately 4 mol %, whereas the minor peak corresponding to Δ5,7,24 accounts for approximately 1.4 mol % of the total sterol.) Both the cholesterol content (8.5 nanomoles/retina) and the total sterol mass (18.3 nanomoles/retina) of treated retinas were significantly lower than the corresponding values for control retinas (25.0 and 26.2 nanomoles/retina, respectively; n = 8; P < 0.01). Control P1 rat brains (Fig. 2C) contained a higher proportion of desmosterol (approximately 28 mol %) than did the corresponding retinas, but cholesterol (approximately 72 mol %) was the dominant sterol, and the HPLC profile was still relatively simple. In contrast, the brain sterol profile of treated P1 animals (Fig. 2D) was more complex; 7-dehydrosterols (approximately 69 mol %) were predominant, withΔ 5,7 accounting for approximately half the total brain sterols and Δ5,7,24 accounting for approximately 20 mol %. Cholesterol also represented approximately 22 mol % of the total, and desmosterol accounted for approximately 7 mol %. Although the liver sterol profile of treated animals (Figs. 2F) was dominated by 7-dehydrocholesterol (approximately 55 mol % of total sterols), appreciable amounts of cholesterol (approximately 39 mol %) and a component with the chromatographic characteristics of desmosterol (approximately 7 mol %) were detected. (The accumulation of the desmosterol-like component is not understood at this time.) 
In contrast, cholesterol represented more than 99% of the total liver sterols in P1 control animals (Fig. 2E) . Squalene levels in P1 control rat livers (approximately 40 ± 22 nanomoles/g) were approximately five times the levels in treated livers (approximately 9 ± 6 nanomoles/g); the squalene levels in other tissues examined were negligible (at or near the level of detection). Control P1 serum (Table 1 ; chromatogram not shown) contained cholesterol as the only identifiable sterol (2.4 micromoles/ml), whereas 25 mol % of the sterols in serum from P1 treated animals was 7-dehydrocholesterol, with the balance accounted for by cholesterol (0.3 micromoles/ml; 12.5% of control value; P < 0.01). 
Phenotypic Features and Retinal Histology and Ultrastructure on P28
Over the ensuing 4-week postnatal course, the treated animals continued to lag behind the control animals in body weight and size. By P28, treated pups were markedly smaller than age-matched control animals (Fig. 3 A), with body weight only approximately 35% of control animals. This aside, the treated animals exhibited a qualitatively normal phenotypic appearance, and notably did not have cataracts or other overt ocular abnormalities (Fig. 3B) . Furthermore, on examination at the light microscopic level, the retinal histology of AY9944-treated animals (Fig. 3D) was indistinguishable from that of the corresponding control animals (Fig. 3C) . Although we performed no quantitative morphometric analyses, qualitative examination of comparable regions of treated and control retinas revealed no obvious differences between the two groups. Also, we found no qualitative evidence of increased cell death, gliosis, or other cytopathologic features in the retinas of treated animals compared with those of control animals. 
Electron microscopic evaluation of P28 retinas from AY9944-treated animals revealed ultrastructural features typical of normal rat retinas at this stage of postnatal development (Fig. 4) . The retinal pigment epithelium (Fig. 4A) exhibited normal morphology, with the usual polarized distribution of intracellular organelles and did not contain any unusual cytoplasmic inclusions. Rod inner segments, ROS, and nuclei also appeared normal (Fig. 4B) . At higher magnification, disc membranes exhibited their normal morphology and highly ordered arrangement in the ROS (Fig. 4C) , consistent with normal photoreceptor morphogenesis. 
Tissue Sterol Composition on P28
Despite the normal appearance of treated retinas at the histologic and ultrastructural levels, sterol composition was grossly different from that of control animals. HPLC analysis of the nonsaponifiable lipids from P28 treated animals (Fig. 5 and Table 1 ) showed that 7-dehydrocholesterol was the predominant sterol in all tissues analyzed, including serum (Fig. 5A) , liver (Fig. 5B) , brain (Fig. 5C) , and neural retina (Fig. 5D) . In these tissues, the mole ratio of 7-dehydrocholesterol to cholesterol (Δ5,75) was serum, 5.0; liver, 5.5; brain, 7.0; and retina, 3.8. 
We also analyzed the nonsaponifiable lipids of ROS membranes from P28 treated animals (data not shown), and found the HPLC profiles to be virtually identical with those of whole neural retina, withΔ 5,75 mole ratio of approximately 3.4. The cholesterol levels in all tissues from treated animals were strikingly reduced (P < 0.01), relative to control animals: retina, 18.5%; brain, 4.2%; liver, 8.5%; and serum, 5.3% (all values expressed as a percentage of control values). In contrast to treated animals, none of these tissues from P28 control animals contained appreciable amounts of 7-dehydrosterols (see Figs. 5E 5F 5G 5H ), and cholesterol was by far the predominant, if not the only, sterol present. 
With specific regard to retina, the total sterol content in the treated group was approximately 89% of the control value (37.4 ± 6.1 nanomoles/retina versus 41.9 ± 4.5 nanomoles/retina). However, this difference was not statistically significant (P > 0.05) and qualitatively can be attributed to the difference in total retinal mass, given the smaller eye size of the treated animals. Although control retinas contained no appreciable 7-dehydrosterols, desmosterol was present at a level of 0.3 ± 0.1 nanomoles/retina, representing less than 1 mol % of the total sterol, with the balance accounted for by cholesterol. With the exception of liver, none of the P28 tissues examined contained appreciable amounts of squalene. This is in good agreement with our previous report that showed no detectable squalene in normal adult rat retina. 15 Also, squalene levels in control livers (54 ± 3 nanomoles/g) were not grossly different from those of treated animals (38 ± 22 nanomoles/g, approximately 70% of control; P > 0.05). 
Effect of AY9944 on Retinal Electrophysiology
It should be noted that normal tissue histology does not necessarily imply normal function. For example, in one animal model of congenital stationary night blindness, 21 retinal morphology is relatively normal, yet electrophysiological function is severely compromised. To evaluate the possible functional consequences of this drastic alteration of retina lipid composition, P28 treated and control animals were examined by electroretinography (Fig. 6) . As shown in Figure 6A 6a series of dark-adapted ERGs was generated for each animal (n = 4 per group). Not only was there no deficit in electrophysiological function in the treated group, those animals actually exhibited larger responses compared with the control animals: Whereas the lowest flash intensity produced only a barely measurable b-wave amplitude in control animals, all treated animals exhibited a clear b-wave response at this stimulus intensity. The response amplitudes of the major ERG components are plotted as a function of stimulus intensity in Figure 6B . The apparent difference in response amplitudes between the treated and control groups was significant for the b-wave (F[1,6] = 7.0; P < 0.05) and for the a-wave (F[1,6] = 6.2; P < 0.05). 
We also evaluated the response kinetics of the ERG a- and b-wave components in the treated and control groups (Fig. 6C) . The implicit times were greater in the treated group compared with control animals; this difference was significant for the b-wave (F[1,6]) = 28.2; P < 0.01), but not for the a-wave (F[1,6] = 4.1; nonsignificant). These results indicate that the G-protein–coupled cascades involved in phototransduction and the rod-depolarizing bipolar cell response, which underlie, respectively, the ERG a- and b-waves, 22 23 are not altered by cholesterol reduction and replacement with 7-dehydrosterols. However, the results are consistent with an overall decline in the deactivation kinetics of these cascades, resulting in larger, but slower, ERG responses. 
Discussion
We have pharmacologically manipulated the sterol composition of the rat retina in the course of embryologic and early postnatal development so that the cholesterol normally present in tissues was largely replaced with 7-dehydrosterols (e.g., 7-dehydrocholesterol). Despite this, treated animals did not display gross teratogenic effects, nor was there any observable derangement in retinal development, histologic organization, ultrastructure, or electrophysiological competence. Therefore, such alterations in lipid metabolism do not necessarily result in dramatic phenotypic defects. 
These findings were surprising, given the previous reports of teratogenic effects of AY9944 in experimental animals, 13 14 and the clinical features associated with the Smith–Lemli–Opitz (SLO) syndrome, 24 25 26 27 a frequently fatal, autosomal recessive disorder characterized by multiple dysmorphic features, profound mental retardation, and failure to thrive. Patients with SLO syndrome exhibit markedly low levels of cholesterol and extraordinarily high levels of 7-dehydrosterols (especially 7-dehydrocholesterol) in all bodily tissues, compared with normal subjects, indicative of a metabolic defect in cholesterol biosynthesis involving the conversion of 7-dehydrosterols to cholesterol. 26 27 However, there is a broad range of variance in the sterol composition of tissues in SLO-affected people, and the levels of 7-dehydrosterol do not necessarily correlate with the severity of the disease. 26 27 The enzyme 3β-hydroxysterol Δ 7 -reductase (EC1.3.1.21) recently has been cloned and shown to be defective in SLO-affected patients. 28 29 30 31  
We also observed that our AY9944-treated rats did not exhibit cataracts, in marked contrast to patients with SLO 32 33 34 35 and to rats that have been treated in a similar manner with hypolipidemic agents such as U18666A, an inhibitor of desmosterol reductase. 36 37 However, the histopathology of the retina observed in our study appears to correlate well with that reported in the sole case study of retinal histopathology in a patient affected by SLO syndrome, 35 which described a 1-month-old boy who had multiple phenotypic and physiological features consistent with SLO syndrome. Remarkably, the retinas exhibited relatively normal histology, with the typical orderly stratification of cell layers and ultrastructurally differentiated rods and cones. These findings suggest that SLO syndrome may not involve gross retinal disease or dysfunction, at least at an early age. The only significant retinal abnormalities (observed in that study, but not in ours) were incipient drop-out of peripheral ganglion cells (with consequent neuronal atrophy in the optic nerve) and the presence of uncharacterized “cytoplasmic masses” in the subretinal space. 
Our histologic and ultrastructural findings also are at variance with an earlier study, 38 which reported the appearance of lamellar inclusion bodies in the retina, lens, and various ocular cells and in the brain, spleen, and liver of rats administered 50 mg/kg AY9944 daily by intraperitoneal injection for 5 days, beginning on P2. That report also claimed that prolonged administration of the compound caused degeneration of the retina and cataractous changes in the lens, neither of which was observed in the present study. It also should be noted that such lamellar inclusions were not observed in the SLO histopathology study reported by Kretzer et al. 35 It is possible that the discrepancies between our study and that of Sakuragawa et al. 38 may have been caused by the considerably higher doses of AY9944 used in their study. In our experience, systemic injection of AY9944 at levels of 50 mg/kg or more (every other day, from P1) leads to severe debilitation and high mortality within 1 week of treatment. 
Rodent models that mimic the biochemical and some of the phenotypic hallmarks of the SLO syndrome have been produced by treatment with 7-dehydrocholesterol Δ7-reductase inhibitors similar to that used in the present study. 39 40 It should be noted that our animal model produced reductions in total serum sterols (approximately 32% of control animals, at P28) and cholesterol levels (approximately 5% of control animals, at P28) and elevation of tissue 7-dehydrocholesterol levels comparable to or greater than that observed in either SLO patients or the previously reported animal models. Those prior studies, however, did not investigate retina sterol composition, histology, or function. Also, those studies used considerably higher doses of drug (up to 300 mg/kg per day) than that used in the present study—doses that may be cytotoxic for reasons other than their inhibition of 3β-hydroxysterol Δ7-reductase. We found that such doses were not required to achieve the biochemical hallmarks of dramatic reduction in tissue cholesterol levels and marked accumulation of 7-dehydrosterols. In addition, it is well known that the timing of administration of AY9944 during gestation is critical, with the highest susceptibility to development of teratogenic effects occurring within the first few days. 14 We specifically chose to administer AY9944 no earlier than the sixth gestational day and at the given dose, because empirically we found that earlier presentation and higher doses resulted in stillbirths. Notably, even in such stillborn animals, there was no evidence of retinal dysplasia or other ocular malformations (data not shown). 
Because we did not observe cytologic abnormalities or electrophysiological defects in AY9944-treated animals, our results suggest that 7-dehydrocholesterol may be able to substitute“ functionally” for cholesterol in the retina. This is surprising, in view of biophysical and biologic studies that would suggest 7-dehydrocholesterol is a poor substitute for cholesterol in certain membrane-dependent functions or activities. 1 5 6 However, it should be noted that those studies involved complete replacement of cholesterol with alternate sterols, such as 7-dehydrocholesterol. This was not the case in the present study, because we were unable to achieve total depletion of cholesterol in the retina or other tissues. Therefore, perhaps there is a threshold level of cholesterol necessary for promotion and preservation of normal retinal histogenesis, below which drastic cytologic effects may occur. Alternatively, perhaps there is some synergism between the abnormally low levels of endogenous cholesterol and the abnormally high levels of 7-dehydrocholesterol that permits normal cell structure and function to be achieved. Such“ sterol synergism” has been demonstrated in procaryotes, 41 lower eukaryotic organisms (e.g., yeast, 42 43 44 and Paramecium 45 ), and vertebrate-derived cells (e.g., LM mouse fibroblasts). 46 In these cases, the accommodation of a nonphysiological sterol typically is accompanied by adaptive changes in the fatty acid profile of membrane phospholipids and also can involve changes in phospholipid class composition and sterol/phospholipid mole ratios. These kinds of adaptive changes in retinal lipid composition remain to be evaluated in the present animal model. 
In some cases, replacement of the normal sterol with nonphysiological sterol structures is tolerated in biologic systems. For example, it has been demonstrated recently 9 that C27 3β-hydroxysterols other than cholesterol, including 7-dehydrocholesterol and desmosterol, can replace cholesterol in vitro in the functional covalent modification of Sonic hedgehog protein, a mammalian homologue of the hedgehog family of signal transduction molecules. 7 Although the exact functions of cholesterol in the retina remain to be defined, a specific requirement for cholesterol in modulating rod photoreceptor signal transduction, through rhodopsin–cholesterol interactions, stabilization of rhodopsin and early photopigment “bleaching” intermediates, and possible membrane structure stabilization, has been suggested by the results of studies by Boesze—Battaglia and Albert 47 and Albert et al. 48 49 Considering our findings, it would be of interest to evaluate the ability of 7-dehydrocholesterol to replace cholesterol in similar studies. 
 
Figure 1.
 
AY9944 treatment does not alter ocular morphogenesis or retinal histogenesis. (A) Vertical cross section of paraffin-embedded P1 control rat eye, stained with hematoxylin-eosin, illustrating normal appearance and spatial relationships of retina (R), lens (L), and cornea (C). (B) Corresponding P1 eye from AY9944-treated rat, exhibiting tissue morphology comparable to that in control animals. (C) Longitudinal section through central retina (posterior pole) of P1 control eye, illustrating early postnatal histologic features of the normal rat retina. (D) Corresponding longitudinal section through central retina of P1 AY9944-treated rat eye, exhibiting similar histologic appearance to control. Scale bar (C, D), 25 μm. Gcl, ganglion cell layer; chor, choroid.
Figure 1.
 
AY9944 treatment does not alter ocular morphogenesis or retinal histogenesis. (A) Vertical cross section of paraffin-embedded P1 control rat eye, stained with hematoxylin-eosin, illustrating normal appearance and spatial relationships of retina (R), lens (L), and cornea (C). (B) Corresponding P1 eye from AY9944-treated rat, exhibiting tissue morphology comparable to that in control animals. (C) Longitudinal section through central retina (posterior pole) of P1 control eye, illustrating early postnatal histologic features of the normal rat retina. (D) Corresponding longitudinal section through central retina of P1 AY9944-treated rat eye, exhibiting similar histologic appearance to control. Scale bar (C, D), 25 μm. Gcl, ganglion cell layer; chor, choroid.
Figure 2.
 
AY9944 treatment produces marked alteration of sterol composition of retina and other rat tissues by P1. Elution positions of authentic standards of cholesterol (Δ5), 7-dehydrocholesterol (Δ5,7), desmosterol (Δ 5 24 ), 7-dehydrodesmosterol (Δ5,7,24), and squalene (Sq) are indicated. Control tissues: (A) retina; (C) brain; (E) liver. AY9944-treated tissues: (B) retina; (D) brain; (F) liver. Note abnormal accumulation of 7-dehydrosterols and relative reduction of cholesterol in AY9944-treated tissues, compared with control animals.
Figure 2.
 
AY9944 treatment produces marked alteration of sterol composition of retina and other rat tissues by P1. Elution positions of authentic standards of cholesterol (Δ5), 7-dehydrocholesterol (Δ5,7), desmosterol (Δ 5 24 ), 7-dehydrodesmosterol (Δ5,7,24), and squalene (Sq) are indicated. Control tissues: (A) retina; (C) brain; (E) liver. AY9944-treated tissues: (B) retina; (D) brain; (F) liver. Note abnormal accumulation of 7-dehydrosterols and relative reduction of cholesterol in AY9944-treated tissues, compared with control animals.
Table 1.
 
Table 1.
 
Sterol Composition of Tissues from Control- and AY9944-Treated Rats*
Table 1.
 
Table 1.
 
Sterol Composition of Tissues from Control- and AY9944-Treated Rats*
Tissue Sterol P1 P28
Control +AY9944 Control +AY9944
Mean ± SD % of Total Mean ± SD % of Total % of Control Mean ± SD % of Total Mean ± SD % of Total % of Control
Retina, †
Δ5 25.0 ± 2.4 95.4 8.5 ± 3.6 46.4 34.0, ∥ 41.6 ± 4.5 99.3 7.7 ± 1.1 20.6 18.5, ∥
Δ5,7 8.6 ± 3.6 47.0 29.4 ± 5.4 78.6
Δ5,24 1.2 ± 0.3 4.6 0.7 ± 0.4 3.8 58.3 0.3 ± 0.1 0.7 0.2 ± 0.1 0.5 66.7
Δ5,7,24 0.5 ± 0.3 2.7 0.1 ± 0.1 0.3
Total 26.2 ± 2.4 18.3 ± 5.2 69.8, ∥ 41.9 ± 4.5 37.4 ± 6.1 89.2
Brain, ‡
Δ5 2.1 ± 0.2 72.4 1.2 ± 0.5 22.2 57.1, ∥ 19.2 ± 7.1 98.5 0.8 ± 0.2 11.3 4.2, ∥
Δ5,7 2.7 ± 0.9 50.0 5.6 ± 0.7 78.9
Δ5,24 0.8 ± 0.1 27.6 0.4 ± 0.1 7.4 50.0, ∥ 0.3 ± 0.0 1.5 0.2 ± 0.1 2.8 66.7
Δ5,7,24 1.1 ± 0.4 20.4 0.5 ± 0.1 7.0
Total 2.9 ± 0.2 5.4 ± 1.9 186, ∥ 19.5 ± 7.1 7.1 ± 0.7 36.4, ∥
Liver, ‡
Δ5 4.1 ± 1.4 100 1.7 ± 0.9 38.6 38.1, ∥ 4.7 ± 0.3 100 0.4 ± 0.2 15.4 8.5, ∥
Δ5,7 2.4 ± 1.5 54.5 2.2 ± 0.8 84.6
Δ5,24 0.3 ± 0.1 6.8
Δ5,7,24
Total 4.1 ± 1.4 4.4 ± 1.3 107 4.7 ± 0.3 2.6 ± 0.8 55.3, ∥
Serum, §
Δ5 2.4 ± 0.2 100 0.3 ± 0.2 75.0 12.5, ∥ 1.9 ± 0.5 100 0.1 ± 0.1 16.7 5.3, ∥
Δ5,7 0.1 ± 0.1 25.0 0.5 ± 0.3 83.3
Total 2.4 ± 0.2 0.4 ± 0.2 16.7, ∥ 1.9 ± 0.5 0.6 ± 0.3 33.6, ∥
Figure 3.
 
AY9944 treatment did not cause dysmorphic features or affect cytologic maturation of retina within the first postnatal month. (A) Gross appearance of P28 control (left) and AY9944-treated rats (right). (B) Left frontal view of AY9944-treated rat, demonstrating absence of cataract. (C) Longitudinal section through central retina (posterior pole) of P28 control eye. Retina is stratified into histologically distinct layers. (D) Corresponding longitudinal section of retina from eye of P28 AY9944-treated rat, exhibiting histologic features comparable to control. Gcl, ganglion cell layer; ipl, inner plexiform layer; inl, inner nuclear layer; opl, outer plexiform layer; onl, outer nuclear layer; ris, rod inner segment layer; ros, rod outer segment layer; rpe, retinal pigment epithelium. Scale bar, (C, D) 25 μm.
Figure 3.
 
AY9944 treatment did not cause dysmorphic features or affect cytologic maturation of retina within the first postnatal month. (A) Gross appearance of P28 control (left) and AY9944-treated rats (right). (B) Left frontal view of AY9944-treated rat, demonstrating absence of cataract. (C) Longitudinal section through central retina (posterior pole) of P28 control eye. Retina is stratified into histologically distinct layers. (D) Corresponding longitudinal section of retina from eye of P28 AY9944-treated rat, exhibiting histologic features comparable to control. Gcl, ganglion cell layer; ipl, inner plexiform layer; inl, inner nuclear layer; opl, outer plexiform layer; onl, outer nuclear layer; ris, rod inner segment layer; ros, rod outer segment layer; rpe, retinal pigment epithelium. Scale bar, (C, D) 25 μm.
Figure 4.
 
Ultrastructural features of P28 retina appear normal in AY9944-treated rats. (A) Electron micrograph of the retinal pigment epithelium (RPE) exhibits normal morphology and distribution of intracellular organelles, including nucleus (n), mitochondria (m), basal infoldings of plasma membrane (bi), and Bruch’s membrane (bm). The tips of rod outer segments (ros) are invaginated into the apical villi (ap) of the RPE. A phagosome (p) derived from a recently shed ROS tip is seen in the early stages of ingestion. (B) Low-magnification view of the photoreceptor layer, showing well-organized rod outer segments (ros) and inner segments (ris) and normal photoreceptor nuclei (n). (C) Higher magnification of rod cells, illustrating normal alignment and ultrastructure of ROS disc membranes (arrowheads) and connecting cilium (ml). Scale bars: (A, B) 2 μm; (C) 0.5 μm.
Figure 4.
 
Ultrastructural features of P28 retina appear normal in AY9944-treated rats. (A) Electron micrograph of the retinal pigment epithelium (RPE) exhibits normal morphology and distribution of intracellular organelles, including nucleus (n), mitochondria (m), basal infoldings of plasma membrane (bi), and Bruch’s membrane (bm). The tips of rod outer segments (ros) are invaginated into the apical villi (ap) of the RPE. A phagosome (p) derived from a recently shed ROS tip is seen in the early stages of ingestion. (B) Low-magnification view of the photoreceptor layer, showing well-organized rod outer segments (ros) and inner segments (ris) and normal photoreceptor nuclei (n). (C) Higher magnification of rod cells, illustrating normal alignment and ultrastructure of ROS disc membranes (arrowheads) and connecting cilium (ml). Scale bars: (A, B) 2 μm; (C) 0.5 μm.
Figure 5.
 
Sterol composition of rat tissues from P28 AY9944-treated rats (A, B, C, D) was markedly different from that of P28 control rats (E, F, G, H). Reversed-phase HPLC profiles (compare Fig. 2 ) illustrate predominance of 7-dehydrocholesterol (Δ5,7) and reduced cholesterol levels in specimens from AY9944-treated rats, whereas control tissues were devoid of 7-dehydrosterols, and cholesterol was the dominant, if not the only, sterol present. Serum (A, E); liver (B, F); neural retina (C, G); brain (D, H).
Figure 5.
 
Sterol composition of rat tissues from P28 AY9944-treated rats (A, B, C, D) was markedly different from that of P28 control rats (E, F, G, H). Reversed-phase HPLC profiles (compare Fig. 2 ) illustrate predominance of 7-dehydrocholesterol (Δ5,7) and reduced cholesterol levels in specimens from AY9944-treated rats, whereas control tissues were devoid of 7-dehydrosterols, and cholesterol was the dominant, if not the only, sterol present. Serum (A, E); liver (B, F); neural retina (C, G); brain (D, H).
Figure 6.
 
AY9944-treated retinas (P28) exhibit electrophysiological function similar to that in control animals. (A) Dark-adapted ERGs from a representative control (left) and an AY9944-treated rat (right). Flash intensities are shown at right. (B) Intensity-response functions for a-wave and b-wave amplitudes from AY9944-treated (•) and control animals (○). Data represent average values ± 1 SD (n = 4). (C) ERG a-wave and b-wave implicit times plotted as a function of stimulus intensity.
Figure 6.
 
AY9944-treated retinas (P28) exhibit electrophysiological function similar to that in control animals. (A) Dark-adapted ERGs from a representative control (left) and an AY9944-treated rat (right). Flash intensities are shown at right. (B) Intensity-response functions for a-wave and b-wave amplitudes from AY9944-treated (•) and control animals (○). Data represent average values ± 1 SD (n = 4). (C) ERG a-wave and b-wave implicit times plotted as a function of stimulus intensity.
This article is dedicated to the memory of George J. Schroepfer, Jr (d. December 11, 1998). The authors thank Kurt Steiner (Wyeth–Ayerst Research) for the generous gift of AY9944; David Nes, Robert A. Pascal, and George J. Schroepfer, Jr, for authentic sterol standards; and R. Kennedy Keller and Gene C. Ness for helpful discussions during the course of this study. 
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Figure 1.
 
AY9944 treatment does not alter ocular morphogenesis or retinal histogenesis. (A) Vertical cross section of paraffin-embedded P1 control rat eye, stained with hematoxylin-eosin, illustrating normal appearance and spatial relationships of retina (R), lens (L), and cornea (C). (B) Corresponding P1 eye from AY9944-treated rat, exhibiting tissue morphology comparable to that in control animals. (C) Longitudinal section through central retina (posterior pole) of P1 control eye, illustrating early postnatal histologic features of the normal rat retina. (D) Corresponding longitudinal section through central retina of P1 AY9944-treated rat eye, exhibiting similar histologic appearance to control. Scale bar (C, D), 25 μm. Gcl, ganglion cell layer; chor, choroid.
Figure 1.
 
AY9944 treatment does not alter ocular morphogenesis or retinal histogenesis. (A) Vertical cross section of paraffin-embedded P1 control rat eye, stained with hematoxylin-eosin, illustrating normal appearance and spatial relationships of retina (R), lens (L), and cornea (C). (B) Corresponding P1 eye from AY9944-treated rat, exhibiting tissue morphology comparable to that in control animals. (C) Longitudinal section through central retina (posterior pole) of P1 control eye, illustrating early postnatal histologic features of the normal rat retina. (D) Corresponding longitudinal section through central retina of P1 AY9944-treated rat eye, exhibiting similar histologic appearance to control. Scale bar (C, D), 25 μm. Gcl, ganglion cell layer; chor, choroid.
Figure 2.
 
AY9944 treatment produces marked alteration of sterol composition of retina and other rat tissues by P1. Elution positions of authentic standards of cholesterol (Δ5), 7-dehydrocholesterol (Δ5,7), desmosterol (Δ 5 24 ), 7-dehydrodesmosterol (Δ5,7,24), and squalene (Sq) are indicated. Control tissues: (A) retina; (C) brain; (E) liver. AY9944-treated tissues: (B) retina; (D) brain; (F) liver. Note abnormal accumulation of 7-dehydrosterols and relative reduction of cholesterol in AY9944-treated tissues, compared with control animals.
Figure 2.
 
AY9944 treatment produces marked alteration of sterol composition of retina and other rat tissues by P1. Elution positions of authentic standards of cholesterol (Δ5), 7-dehydrocholesterol (Δ5,7), desmosterol (Δ 5 24 ), 7-dehydrodesmosterol (Δ5,7,24), and squalene (Sq) are indicated. Control tissues: (A) retina; (C) brain; (E) liver. AY9944-treated tissues: (B) retina; (D) brain; (F) liver. Note abnormal accumulation of 7-dehydrosterols and relative reduction of cholesterol in AY9944-treated tissues, compared with control animals.
Figure 3.
 
AY9944 treatment did not cause dysmorphic features or affect cytologic maturation of retina within the first postnatal month. (A) Gross appearance of P28 control (left) and AY9944-treated rats (right). (B) Left frontal view of AY9944-treated rat, demonstrating absence of cataract. (C) Longitudinal section through central retina (posterior pole) of P28 control eye. Retina is stratified into histologically distinct layers. (D) Corresponding longitudinal section of retina from eye of P28 AY9944-treated rat, exhibiting histologic features comparable to control. Gcl, ganglion cell layer; ipl, inner plexiform layer; inl, inner nuclear layer; opl, outer plexiform layer; onl, outer nuclear layer; ris, rod inner segment layer; ros, rod outer segment layer; rpe, retinal pigment epithelium. Scale bar, (C, D) 25 μm.
Figure 3.
 
AY9944 treatment did not cause dysmorphic features or affect cytologic maturation of retina within the first postnatal month. (A) Gross appearance of P28 control (left) and AY9944-treated rats (right). (B) Left frontal view of AY9944-treated rat, demonstrating absence of cataract. (C) Longitudinal section through central retina (posterior pole) of P28 control eye. Retina is stratified into histologically distinct layers. (D) Corresponding longitudinal section of retina from eye of P28 AY9944-treated rat, exhibiting histologic features comparable to control. Gcl, ganglion cell layer; ipl, inner plexiform layer; inl, inner nuclear layer; opl, outer plexiform layer; onl, outer nuclear layer; ris, rod inner segment layer; ros, rod outer segment layer; rpe, retinal pigment epithelium. Scale bar, (C, D) 25 μm.
Figure 4.
 
Ultrastructural features of P28 retina appear normal in AY9944-treated rats. (A) Electron micrograph of the retinal pigment epithelium (RPE) exhibits normal morphology and distribution of intracellular organelles, including nucleus (n), mitochondria (m), basal infoldings of plasma membrane (bi), and Bruch’s membrane (bm). The tips of rod outer segments (ros) are invaginated into the apical villi (ap) of the RPE. A phagosome (p) derived from a recently shed ROS tip is seen in the early stages of ingestion. (B) Low-magnification view of the photoreceptor layer, showing well-organized rod outer segments (ros) and inner segments (ris) and normal photoreceptor nuclei (n). (C) Higher magnification of rod cells, illustrating normal alignment and ultrastructure of ROS disc membranes (arrowheads) and connecting cilium (ml). Scale bars: (A, B) 2 μm; (C) 0.5 μm.
Figure 4.
 
Ultrastructural features of P28 retina appear normal in AY9944-treated rats. (A) Electron micrograph of the retinal pigment epithelium (RPE) exhibits normal morphology and distribution of intracellular organelles, including nucleus (n), mitochondria (m), basal infoldings of plasma membrane (bi), and Bruch’s membrane (bm). The tips of rod outer segments (ros) are invaginated into the apical villi (ap) of the RPE. A phagosome (p) derived from a recently shed ROS tip is seen in the early stages of ingestion. (B) Low-magnification view of the photoreceptor layer, showing well-organized rod outer segments (ros) and inner segments (ris) and normal photoreceptor nuclei (n). (C) Higher magnification of rod cells, illustrating normal alignment and ultrastructure of ROS disc membranes (arrowheads) and connecting cilium (ml). Scale bars: (A, B) 2 μm; (C) 0.5 μm.
Figure 5.
 
Sterol composition of rat tissues from P28 AY9944-treated rats (A, B, C, D) was markedly different from that of P28 control rats (E, F, G, H). Reversed-phase HPLC profiles (compare Fig. 2 ) illustrate predominance of 7-dehydrocholesterol (Δ5,7) and reduced cholesterol levels in specimens from AY9944-treated rats, whereas control tissues were devoid of 7-dehydrosterols, and cholesterol was the dominant, if not the only, sterol present. Serum (A, E); liver (B, F); neural retina (C, G); brain (D, H).
Figure 5.
 
Sterol composition of rat tissues from P28 AY9944-treated rats (A, B, C, D) was markedly different from that of P28 control rats (E, F, G, H). Reversed-phase HPLC profiles (compare Fig. 2 ) illustrate predominance of 7-dehydrocholesterol (Δ5,7) and reduced cholesterol levels in specimens from AY9944-treated rats, whereas control tissues were devoid of 7-dehydrosterols, and cholesterol was the dominant, if not the only, sterol present. Serum (A, E); liver (B, F); neural retina (C, G); brain (D, H).
Figure 6.
 
AY9944-treated retinas (P28) exhibit electrophysiological function similar to that in control animals. (A) Dark-adapted ERGs from a representative control (left) and an AY9944-treated rat (right). Flash intensities are shown at right. (B) Intensity-response functions for a-wave and b-wave amplitudes from AY9944-treated (•) and control animals (○). Data represent average values ± 1 SD (n = 4). (C) ERG a-wave and b-wave implicit times plotted as a function of stimulus intensity.
Figure 6.
 
AY9944-treated retinas (P28) exhibit electrophysiological function similar to that in control animals. (A) Dark-adapted ERGs from a representative control (left) and an AY9944-treated rat (right). Flash intensities are shown at right. (B) Intensity-response functions for a-wave and b-wave amplitudes from AY9944-treated (•) and control animals (○). Data represent average values ± 1 SD (n = 4). (C) ERG a-wave and b-wave implicit times plotted as a function of stimulus intensity.
Table 1.
 
Table 1.
 
Sterol Composition of Tissues from Control- and AY9944-Treated Rats*
Table 1.
 
Table 1.
 
Sterol Composition of Tissues from Control- and AY9944-Treated Rats*
Tissue Sterol P1 P28
Control +AY9944 Control +AY9944
Mean ± SD % of Total Mean ± SD % of Total % of Control Mean ± SD % of Total Mean ± SD % of Total % of Control
Retina, †
Δ5 25.0 ± 2.4 95.4 8.5 ± 3.6 46.4 34.0, ∥ 41.6 ± 4.5 99.3 7.7 ± 1.1 20.6 18.5, ∥
Δ5,7 8.6 ± 3.6 47.0 29.4 ± 5.4 78.6
Δ5,24 1.2 ± 0.3 4.6 0.7 ± 0.4 3.8 58.3 0.3 ± 0.1 0.7 0.2 ± 0.1 0.5 66.7
Δ5,7,24 0.5 ± 0.3 2.7 0.1 ± 0.1 0.3
Total 26.2 ± 2.4 18.3 ± 5.2 69.8, ∥ 41.9 ± 4.5 37.4 ± 6.1 89.2
Brain, ‡
Δ5 2.1 ± 0.2 72.4 1.2 ± 0.5 22.2 57.1, ∥ 19.2 ± 7.1 98.5 0.8 ± 0.2 11.3 4.2, ∥
Δ5,7 2.7 ± 0.9 50.0 5.6 ± 0.7 78.9
Δ5,24 0.8 ± 0.1 27.6 0.4 ± 0.1 7.4 50.0, ∥ 0.3 ± 0.0 1.5 0.2 ± 0.1 2.8 66.7
Δ5,7,24 1.1 ± 0.4 20.4 0.5 ± 0.1 7.0
Total 2.9 ± 0.2 5.4 ± 1.9 186, ∥ 19.5 ± 7.1 7.1 ± 0.7 36.4, ∥
Liver, ‡
Δ5 4.1 ± 1.4 100 1.7 ± 0.9 38.6 38.1, ∥ 4.7 ± 0.3 100 0.4 ± 0.2 15.4 8.5, ∥
Δ5,7 2.4 ± 1.5 54.5 2.2 ± 0.8 84.6
Δ5,24 0.3 ± 0.1 6.8
Δ5,7,24
Total 4.1 ± 1.4 4.4 ± 1.3 107 4.7 ± 0.3 2.6 ± 0.8 55.3, ∥
Serum, §
Δ5 2.4 ± 0.2 100 0.3 ± 0.2 75.0 12.5, ∥ 1.9 ± 0.5 100 0.1 ± 0.1 16.7 5.3, ∥
Δ5,7 0.1 ± 0.1 25.0 0.5 ± 0.3 83.3
Total 2.4 ± 0.2 0.4 ± 0.2 16.7, ∥ 1.9 ± 0.5 0.6 ± 0.3 33.6, ∥
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