Ultrastructural abnormalities of epididymal tissues in XXSxr pseudomale (sex-reversed) mice.

Sex reversed (Sxr), a duplication of the Y chromosomal testis-determining factor in mice, causes testis development in XXSxr animals. No effects of Sxr on nongonadal organs are expected. However, we have previously shown that the epididymis of XXSxr pseudomale (sex-reversed) mice lacks the Initial Segment. In the present study we examined the ultrastructure of the head of the epididymis of adult and 21-day old XXSxr pseudomale mice. Epithelial cells of both adult and juvenile XXSxr animals contain numerous vesicles, some within mitochondria. The basal lamina is thickened and infolded. The periepithelial layer is abnormally thick, with distorted smooth muscle cells and fibrocytes that also contain lysosomelike vesicles, often in mitochondria, and excessively wide intercellular spaces. Normal collagen fibrils are infrequent; they are in part replaced by wisps of nondiscrete material, possibly immature collagen. Sxr is known to affect spermatogenesis and Sertoli cells. The abnormal conjugation of sex-determining genes in XXSxr appears also to subvert mesenchymal-epithelial development in both epididymis and testis. We believe the most likely explanation of our data is that the XXSxr genotype is not testis specific but also influences the epididymis directly.

Connective tissue metabolism in muscular dystrophy. Early amino acid changes in collagen types isolated from the gastrocnemius muscle of developing dystrophic chicken embryos.

The amino acid composition of all collagen types present in the gastrocnemius muscle of dystrophic chick embryos showed an altered profile at both day 14 and day 20 in ovo when compared with the controls. The changes observed at both day 14 and day 20 in ovo suggests that there is a removal of polar side-chains in dystrophic collagen and substitution with non-polar amino acids. The amino acid composition data between day 14 and day 20 indicated: (a) a decrease in hydroxylation (hydroxyproline and hydroxylysine) with a concurrent increase in proline and lysine and a decrease in the levels of arginine; (b) the levels of glycine and alanine did not change with age; and (c) the ratios of glycine to hydroxyproline and proline to hydroxyproline changed significantly in all dystrophic collagen types between day 14 and day 20. Contrast analysis results clearly showed that the changes in amino acid composition observed in each dystrophic type of collagen between day 14 and day 20 were not due to the effect of aging but to some other factor(s). This study provides more evidence that a problem lies in the biosynthesis of collagen present in developing muscles of dystrophic chick embryos, particularly with respect to the transcription or translation of procollagen genes and/or a failure in the processing and differentiation of collagen types.

Connective tissue metabolism in muscular dystrophy. Amino acid composition of native types I, III, IV and V collagen isolated from the gastrocnemius muscle of embryonic chickens with genetic muscular dystrophy.

The amino acid composition data on types I, III, IV and V collagen isolated from embryonic dystrophic skeletal muscle strongly indicate that alterations in collagen synthesis occur in intramuscular connective tissue of developing muscles in embryonic dystrophic chickens. The changes observed in the amino acid composition of dystrophic collagen were: (a) a selective removal of polar amino acids and substitution with non-polar amino acids; (b) significant decreases in basic (lysine, hydroxylysine and arginine) and hydroxylated (4-hydroxyproline and hydroxylysine) amino acids; and (c) significant increases in the amounts of glycine, proline and alanine. The amino acid substitutions suggest a genetic alteration in the collagen synthesizing process and a change in its structure. The variations in amino acid composition of collagen from dystrophic chickens could give rise to a decrease in both inter- and intramolecular cross-linking, thus decreasing the stability and functionality of newly formed collagen fibrils. The differences associated with the dystrophic collagen reported in this study are probably due to the differences in primary structure in terms of amino acid sequence rather than post-translational modifications. The structural differences noted would also lead to an alteration of the role collagen plays in regulating the differentiation of developing muscles. The changes in amino acid structure strongly suggest that the 'collagen' formed by dystrophic chickens should be considered a collagen-like protein or 'collagenoid'.

Ultrastructure of the developing myotendinous junction of genetic dystrophic chickens.

This paper presents an ultrastructural study of the changes occurring within the tendon and at the insertion of the developing gastrocnemius of White Leghorn chickens and the dystrophic lines 413 and 423 from Davis, CA. The study revealed that the Davis lines contained abnormalities in these areas as early as 13 d in ovo with progressive deterioration to 19 d in ovo. The fibroblasts, collagen, and myofibers showed significant alterations as early as 13 in ovo. Fibroblasts contained abnormal mitochondria and altered Golgi bodies. At day 19, many were ruptured. The average diameter of collagen fibrils was smaller in the dystrophic chickens and myofibers showed a variety of alterations, some of which were severe. The details of these alterations are described and their possible relationship with the etiology of genetic muscular dystrophy is discussed.

Basal lamina changes during tissue interactions in hair follicles--an in vitro study of normal dermal papillae and vitamin A-induced glandular morphogenesis.

Skin pieces from 14-day fetal mice were cultivated for 1-10 days prior to fixation and sectioning. Subsequently, sections were studied by light and transmission electron microscopy. In a standard medium the lateral hair follicle walls showed progressive maturation of the basal lamina, while the hair matrix, at the time of a known tissue interaction, showed the formation of gaps in the basal lamina, with heterotypic cell contacts through the gaps. In a vitamin-A enriched medium similar changes occurred, not only at the hair matrix, but also at lateral follicle walls, at the sites of, and prior to, budding and glandular morphogenesis. This study shows that the induction of hair matrix by dermal papilla may perhaps be added to the list of normal tissue interactions in which heterotypic cell contacts occur. It also suggests that vitamin-A induced glandular morphogenesis might come about through a mechanism resembling a normal tissue interaction.

Collagen levels in tissues from selenium deficient ducks.

1. Nutritional myopathy was produced in ducks by feeding the birds selenium deficient diets. The myopathy increased in severity the longer the ducks were fed the selenium deficient diet. 2. There was less total collagen and lesser amounts of soluble collagen found in the tendons of ducks fed the selenium-deficient diet. Likewise, the collagen fibrils were of decreased diameter in those birds. 3. The fibroblasts present in the tendons of the deficient ducks were degenerate. It appears the selenium deficiency produced damaged fibroblast membranes which resulted in decreased collagen synthesis. The resultant loss of collagen structure could have produced a functional tendotomy which caused myodegeneration.

Electron density distribution of dry avian tendon.

The electron density profile along the unit cell of dry native collagen is determined. The method used is a modification of one that has been used successfully for wet native collagen. Uranyl acetate and phosphotungstic acid were used for isomorphous addition in phase determination and were located by electron microscopy. Structure factor magnitudes for dry collagen with and without the heavy metals were obtained from X-ray diffraction data. The first 9 orders were investigated. Standard Argand diagrams provided a unique solution for the phase of each. The resultant profile suggests that significant conformation changes occur in both the non-helical ends and the triple-helix portions of the molecules.

Ultrastructural studies on the gastrocnemius tendon of selenium-deficient ducklings.

This paper presents an ultrastructural study of the changes occurring in the gastrocnemius tendon of normal and selenium-deficient ducklings from 1 to 12 days of age. The study revealed that the normal 1--8-day-old duckling tendon contained 2 morphologically distinct cell types and a third occurring at Days 8--12. The location of these cells within the endotendineum and fiber bundles and their morphologic characteristics are presented. Most cells showed cytologic alterations as early as 4 days on the deficient diet with progressive deterioration to Day 12. The various cell types showed a differential response to the deficiency. Changes included altered endoplasmic reticulum and Golgi apparatus and eventual cell rupture. The significance of these observations and their possible relationship to the etiology of selenium-deficiency-induced dystrophy is discussed.

Ultrastructural studies of the myotendonous junction of selenium-deficient ducklings.

An ultrastructural study was made of the changes occurring within the gastrocnemius insertion of normal and selenium-deficient ducklings from 1 to 12 days of age. The cytologic characteristics of the fibroblasts, vessels, collagen, and muscle cells are described. Those exposed to the selenium deficiency showed major alterations of all components. The fibroblasts showed changes ranging from collapsed cisternae and degenerating mitochondria to rupture. The capillary endothelium was abnormal, as was the smooth muscle of arteriolar walls. The collagen sizes were altered, and the muscle cell termini showed major pathologic changes. The above alterations occurred within 4 days of exposure of the deficiency. The muscle cells of the body portion of the gastrocnemius showed no alterations until Day 8. The observations present evidence that indicates that connective-tissue-vascular abnormalities precede myopathic changes in nutritionally induced dystrophy. The significance of these findings is discussed with respect to the etiology of nutritionally induced dystrophy.

Skin collagen has an unusual d-spacing.

Small angle X-ray diffraction patterns show a d-spacing of 65 +/- 0.5 nm for the collagen in wet intact skin of amphibian larvae (tadpole) as well as from that of nature frogs, chickens and mice. The collagen in these tissues is largely collagen I which exhibits a d-spacing of 67 +/- 0.5 nm in wet intact unstretched tendons. The d-spacing of the skin collagen did not decrease on drying, while it is well known that that of tendon collagen does decrease on drying. The reasons for the decreased d-spacing in the normal skin are not known but we suggest that the different glycosaminoglycan content of skin may be an important factor.

Experimental confirmation of calculated phases and electron density profile for wet native collagen.

An experimental procedure is developed to phase the reflections obtained in x-ray diffraction investigations of collagen in native wet tendons. Phosphotungstic acid was used for isomorphous addition in phase determination and was located by electron microscopy. Structure factors (with phases) were obtained from the electron microscopy data for the heavy metal. Structure-factor magnitudes for collagen with and without the heavy metal were obtained from the x-ray diffraction data. The first 10 orders were investigated. Standard Argand diagrams provided two solutions for each of these, except the weak sixth order. In each case, one of the two possible solutions agrees well with the phases proposed on theoretical grounds by Hulmes et al. The present results suggest that their other proposed phases are probably correct. An electron density profile along the unit cell of the fibril is presented that shows a distinct step, as expected on the basis of the hole-overlap model. The overlap region is 48% of the length of the unit cell.

Ciliated and secretory epidermis produced from embryonic mammalian skin in organ culture by vitamin A.

When skin from the upper lip of 12-day embryonic mice was grown for ten days in organ culture with 5.7 mug retinol added per ml of biological medium, keratinization was suppressed and a ciliated and secretory epithelium was produced. Ultrastructural features of this epithelium are described. At this very early stage mouse epidermis is thus similar of chick epidermis in its ability to undergo radical metaplasia in response to vitamin A.

Alleviating mortality associated with a vitamin E-selenium deficiency by dietary ascorbic acid.

Adding ascorbic acid to a practical ration deficient in vitamin E and selenium for the growing duck substantially reduced associated mortality. The continued appearance of various myopathies but absence of vascular faults supported implication of a reduced de novo ascorbate synthesis as part of the syndrome. Presumably, alleviation of this induced secondary inadequacy with its more lethal pathology was the primary reason for the lower death rate.

Ultrastructural changes in muscular dystrophy. I. Cardiac tissue of piglets deprived of vitamin E and selenium.

The ultrastructure of cardiac tissue from neonate, 3-, 6-, 8-, and 12-week-old piglets, born of vitamin E- and selenium-deprived sows was studied. A progression of lesions occurred in nonmuscular components of this tissue; the first lesion appeared in connective tissue elements. Fibroblasts and the extracellular compartment appeared most severely altered in the neonate, and progressive vascular damage was very evident from 3 to 12 weeks of age. Similarly, neuronal elements appear altered at 3 weeks and were almost nonevident in areas showing marked lesions at 8 and 12 weeks. Fairly extensive alterations were evident in all of these elements before any marked changes become evident in the muscle. The relevance of these observations is discussed in relation to the etiology of the disease.