James R. Mertz

Retinoic acid signals the direction of ocular elongation in the guinea pig eye

A growing eye becomes myopic after form deprivation (FD) or compensates for the power and sign of imposed spectacle lenses. A possible mediator of the underlying growth changes is all-trans retinoic acid (RA). Eye elongation and refractive error (RE) was manipulated by raising guinea pigs with FD, or a spectacle lens worn on one eye. We found retinal-RA increased in myopic eyes with accelerated elongation and was lower in eyes with inhibited elongation. RA levels in the choroid/sclera combined mirrored these directional changes. Feeding RA (25 mg/kg) repeatedly to guinea pigs, also resulted in rapid eye elongation (up to 5 times normal), and yet the RE was not effected. In conclusion, RA may act as a signal for the direction of ocular growth.

Localization of retinoid binding proteins, retinoid receptors, and retinaldehyde dehydrogenase in the chick eye.

Retinoids have many functions in the eye, including, perhaps, the visual guidance of ocular growth. Therefore, we identified where retinoid receptors, binding proteins, and biosynthetic enzymes are located in the ocular tissues of the chick as a step toward discovering where retinoids are generated and where they act. Using antibodies to interphotoreceptor retinoid binding protein (IRBP), cellular retinol binding protein (CRBP), cellular retinoic acid binding protein (CRABP), cellular retinaldehyde binding protein (CRALBP), retinaldehyde dehydrogenase (RALDH), and retinoic acid receptors (RAR and RXR), we localized these proteins to cells in the retina, retinal pigmented epithelium, choroid and sclera of the chick eye. IRBP was detected in the photoreceptor layer and pigmented epithelium; CRBP was in the pigmented epithelium; CRABP was in amacrine and bipolar cells in the retina; CRALBP was in Müller cells, pigmented epithelium, choroid, and fibrous sclera; RALDH was in retinal amacrine cells, pigmented epithelium, and choroid; RAR was in amacrine cells, choroid, and chondrocytes and fibroblasts in the sclera; and RXR was in amacrine and ganglion cells, bipolar cell nuclei, choroid, and chondrocytes. We also found that the growth-modulating toxins colchicine and quisqualate destroyed selectively different subsets of CRABP-containing amacrine cells. We conclude that the distribution of proteins involved in retinoid metabolism is consistent with a role of retinoids not only in phototransduction, but also in maintenance of cellular phenotype and visual guidance of ocular growth.

Substrate specificities and 13-cis-retinoic acid inhibition of human, mouse and bovine cis-retinol dehydrogenases

Recent studies of the human, mouse and bovine genes for 11-cis-retinol dehydrogenase (11cRDH) and human and mouse 9-cis-retinol dehydrogenase (9cRDH) suggest that they are homologs of the same enzyme. This conclusion is inconsistent with earlier literature indicating that 11cRDH is expressed solely in the eye and does not utilize 9-cis-retinol as a substrate. We have compared directly the kinetic properties of recombinant human and mouse 9cRDH with those of bovine 11cRDH for 9-cis- and 11-cis-retinol and investigated the inhibitory properties of 13-cis-retinoic acid on each of these enzymes. Human and mouse 9cRDH and bovine 11cRDH have very similar kinetic properties towards 9-cis- and 11-cis-retinol oxidation and they respond identically to 13-cis-retinoic acid inhibition. Our biochemical data are consistent with the conclusion that 9cRDH and 11cRDH are the same enzyme.

Purification, partial characterization, and localization of Sak57, an acidic intermediate filament keratin present in rat spermatocytes, spermatids, and sperm

We have purified a 57 kDa protein (designated Sak57, for spermatogenic cell/sperm‐associated keratin) from sodium dodecyl sulfate‐β‐mercaptoethanol(SDS‐βME)‐dissociated outer dense fibers isolated from rat sperm tails. Internal protein sequence analysis of Sak57 yielded two 15‐mer and 10‐mer fragments with 70–100% homology to human, rat, and mouse keratins and corresponding to the 1A and 2A regions of the α‐helical rod domain of keratins. A multiple antigenic peptide (MAP) was constructed using the 10‐mer amino acid sequence KAQYEDIAQK (corresponding to the 2A region) and used as antigen for the production of polyclonal antibodies in rabbit. Anti‐MAP sera were used for further analysis of the biochemical characteristics of Sak57 in testis and sperm tails using chromatofocusing, immunobloting, and [32P]orthophosphate‐labeling. We have found that rat testis displays two immunoreactive proteins: a soluble 83 kDa protein with pl range 5.9–6.3, regarded as a precursor, and both detergent‐insoluble and soluble 57 kDa protein with pl range 5.0–5.9, corresponding to the mature form Sak57. The testicular soluble form was phosphorylated. Rat sperm tail samples displayed only the Sak57 detergent‐insoluble form and its pl was more acidic (4.7–4.8). Whole‐mount electron microscopy of negatively stained preparations of sperm‐derived Sak57 resuspended in SDS‐βME revealed a rod‐shaped pattern. A decrease in the concentration of SDS‐βME resulted in the side‐by‐side aggregation of rod‐shaped Sak57 forming thick bundles. Indirect immunofluorescence was used to determine the localization of Sak57 in isolated outer dense fibers, epididymal sperm, spermatids, and pachytene spermatocytes. Confocal laser scanning microscopy was used to analyze the three‐dimensional arrangement of Sak57 in pachytene spermatocytes. Isolated outer dense fiber and sperm tails displayed an immunoreactive product in the form of linear clusters. In elongating spermatids (steps 10–11), Sak57 immunoreactivity was predominant in the head region whereas pachytene spermatocytes displayed a cortical cytoplasmic distribution. Results of this study demonstrate that Sak57 has the characteristics of a keratin intermediate filament and is present during meiotic and postmeiotic stages of spermatogenesis.