Immunohistochemical detection of bovine ruminal carbonic anhydrase isoenzyme

Summary Rabbits immunized with low-activity ruminal carbonic anhydrase (RCA) isoenzyme, extracted from ruminal epithelial cells isolated by digestion with trupsin, yielded anti-RCA sera which reacted specifically with bovine RCA in double agar gel diffusion and immunoelectrophoretic tests, but failed to cross-react with bovine erythrocyte CA. The localization of RCA was identified in histological sections and isolated ruminal epithelial cell preparations by indirect immunofluorescence and immunoperoxidase tests as the basal, spinosum and granulosum layers of ruminal mucous epithelium.     

Fine structure of the filiform papilla of beagle dogs

Light and electron microscopic examination of the dorsal lingual epithelium of beagle dogs (Canis domesticus) revealed three different regions: that anterior to the filiform papillae, that posterior to the papillae, and an interpapillary region. Whereas the basal and suprabasal cells are similar throughout, differences characterize the intermediate and surface layers. Keratohyalin granules are common in the intermediate layers in the anterior and interpapillary regions, tonofibrils are prominent in the posterior region, and no keratohyalin granules occur. The surface layer of the interpapillary region is not keratinized, that of the anterior region shows soft keratinization, and that of the posterior region shows hard keratinization. The perimeter of keratohyalin granules is composed of ribosomes 10-20 nm in diameter.     

Changes in the activity of alcohol dehydrogenase isoenzymes during the estrus cycle in the vagina of the rat

In rodents, the vaginal epithelium undergoes cyclical changes with an alternating pattern of keratinization and mucification. It has been known for decades that vitamin A and its active form retinoic acid are responsible for normal epithelial homeostasis. However, it has not so far been certain which enzymes catalyze the first and rate-limiting step in retinoic acid synthesis. By means of microdissection and ultrathin-layer gel electrophoresis, alcohol dehydrogenase isoenzyme activity was determined quantitatively in the various layers of the vaginal mucous membrane. It was found that, in the rat, only alcohol dehydrogenase 3 and 4 are expressed. Marked cyclical changes of alcohol dehydrogenase 4 activity in the stratum germinativum of the vaginal epithelium strongly support the assumption that this isoenzyme is responsible for retinoic acid synthesis, and that it is essential for the changes accompanying keratinization and mucification.     

Ultrastructural studies of the transitional zone in the nasopharyngeal epithelium, with special reference to the keratinizing process in the mouse

In the nasopharynx of the SMA mouse, the 'intermediate epithelium' occupies the transitional zone between the ciliated columnar and the stratified squamous epithelia. The intermediate epithelium showed gradations ranging from ciliated stratified low-columnar through stratified cuboidal to stratified squamous type. It is suggested that the intermediate epithelium shows the various stages of the epithelium transforming from the ciliated columnar to the stratified squamous epithelium, and that the basal cells of the ciliated columnar epithelium serve as the germinal layer for the transformation. The intermediate epithelium containing a few keratohyalin granules and many membrane-coating granules represented earlier stages of keratinization. The width of the microprojections in the stratified squamous epithelium was about doubled compared to that in the intermediate epithelium. It is suggested that the difference in width is caused by cell membrane distortion associated with keratinization and is regarded as an important marker of the start of keratinization.     

The effect of atrophy, hyperplasia, and keratinization accompanying the estrous cycle on Langerhans' cells in mouse vaginal epithelium

The morphology and numbers of Langerhans' cells vary in epithelia with different patterns of hyperplasia and keratinization. Langerhans' cells stained for ATPase were compared at five phases of the estrous cycle in murine vaginal epithelium. The cells were more dendritic and sparsely distributed with hyperplasia and were less dendritic and more densely distributed with atrophy. Greater numbers of the cells did not accompany keratinization at estrus. Ultrastructurally, three types of Langerhans' cells were discriminated. The first type, active in protein synthesis and phagocytosis, was commonest in sloughing and atrophic epithelium. The second type, containing accumulated and dispersed, electron-dense bodies presumed to be lysosomes, predominated in hyperplastic epithelium. The third, a mature resting cell, was found only after keratinization was complete. This study shows that Langerhans' cells in murine vaginal epithelium vary in morphology and numbers with the epithelial changes of the estrous cycle which may relate to their immunological role, but does not support the contention that their distribution is important for keratinization.     

Age-dependent sensitivity of basal cells in mouse vaginal epithelium towards the diferentiating action of estrogen.

Following ovariectomy, the vaginal epithelium of the mouse is reduced to two layers of cells, the basal layer that constitutes the germinative compartment and surface layer. Under estrogenic influence, this tissue undergoes keratinization at the expense of asal cell only. The initially superficial cells are progressively sloughed, without taking part in the cornification process. We have shown previously that within the first 12 hours after estrogen administration (that is, before any change in mitotic activity is detected) an intermediate layer of cells is formed. Thurs, a migratory process is induced, which leads to a very rapid redistribution to basal cells into two layers...     

Studies on physiology & biochemistry of female genital tract: response of cervix & vagina of albino rats to estrogen, progesterone & testosterone

Albino rats were administered estogen, progesterone or testosterone daily for 4 weeks to study the ponderal, histological and biochemical changes in the cervix and vagina. Stimulatory signs with increased weight, thickening, keratinization and desquamation of the surface epithelium, loose and edematous stroma were descernible in both the organs under estrogen and to some extent under testosterone but not under progesterone. However, progesterone altered the squamous type of vaginal epithelium to thin layers made up of uboidal cells. Metaplastic changes characterized the epithelium under testosterone. Biochemical responses of the 2 organs were often found to differ quantitatively and qualitatively under identical hormonal conditions. Statistical analyses are presented. Possible reasons for the differential behavior of the 2 organs are discussed.     

Avian scale development. X. Dermal induction of tissue-specific keratins in extraembryonic ectoderm

Epidermal-dermal tissue interactions regulate morphogenesis and tissue-specific keratinization of avian skin appendages. The morphogenesis of scutate scales differs from that of reticulate scales, and the keratin polypeptides of their epidermal surfaces are also different. Do the inductive cues which initiate morphogenesis of these scales also establish the tissue-specific keratin patterns of the epidermis, or does the control of tissue-specific keratinization occur at later stages of development? Unlike feathers, scutate and reticulate scales can be easily separated into their epidermal and dermal components late in development when the major events of morphogenesis have been completed and keratinization will begin. Using a common responding tissue (chorionic epithelium) in combination with scutate and reticulate scale dermises, we find that these embryonic dermises, which have completed morphogenesis, can direct tissue-specific statification and keratinization. In other words, once a scale dermis has acquired its form, through normal morphogenesis, it is no longer able to initiate morphogenesis of that scale, but it can direct tissue-specific stratification and keratinization of a foreign ectodermal epithelium, which itself has not undergone scale morphogenesis.     

Ultrastructural localization of acid phosphatase in nonhuman primate vaginal epithelium

Summary The vagina of the rhesus monkey is lined by a stratified squamous epithelium. However, little is known regarding the cytochemical composition of its cell organelles and the substances found in the intercellular spaces. In this study we have examined the ultrastructural distribution of acid phosphatase in the vaginal epithelium. In basal and parabasal cells reaction product was found in some Golgi cisternae and vesicles and in a variety of cytoplasmic granules. Reaction product was also found in some, but not all, membrane-coating granules. In the upper layers of the epithelium, the membrane-coating granules extruded their contents and acid phosphatase was localized in the intercellular spaces. The possible roles of acid phosphatase in keratinization, desquamation, or modification of substances in the intercellular compartment are discussed.     

Structures Indicative of Keratinization in Lactating Cells of Bovine Mammary Gland

Keratohyalin granule-like aggregates and tangles of filaments similar to epidermal prekeratin fibrils have been observed in lactating cells of bovine mammary epithelium. The concomitant occurrence of such structures, which are characteristic of early stages of keratinization, with typical secretory products such as casein micelles demonstrates that keratinization can take place in functional secretory cells. The observations are discussed in relation to current concepts of keratinization in epidermal and in non-epidermal epithelia.     

Expression of specific keratin markers by rabbit corneal, conjunctival, and esophageal epithelia during vitamin A deficiency

Using an in vivo rabbit model system, we have studied the morphological and biochemical changes in corneal, conjunctival, and esophageal epithelia during vitamin A deficiency. Light and electron microscopy showed that the three epithelia undergo different degrees of morphological keratinization. Corneal and conjunctival epithelia became heavily keratinized, forming multiple layers of superficial, anucleated cornified cells. In contrast, esophageal epithelium underwent only minor morphological changes. To correlate morphological alterations with the expression of specific keratin molecules, we have analyzed the keratins from these epithelia by the immunoblot technique using the subfamily-specific AE1 and AE3 monoclonal antikeratin antibodies. The results indicate that during vitamin A deficiency, all three epithelia express an AE1-reactive, acidic 56.5-kd keratin and an AE3-reactive, basic 65-67-kd keratin. Furthermore, the expression of these two keratins correlated roughly with the degree of morphological keratinization. AE2 antibody (specific for the 56.5- and 65-67-kd keratins) stained keratinized corneal epithelial sections suprabasally, as in the epidermis, suggesting that these two keratins are expressed mainly during advanced stages of keratinization. These two keratins have previously been suggested to represent markers for epidermal keratinization. Our present data indicate that they can also be expressed by other stratified epithelia during vitamin A deficiency-induced keratinization, and suggest the possibility that they may play a role in the formation of the densely packed tonofilament bundles in cornified cells of keratinized tissues.     

Transport of ketone bodies and lactate in the sheep ruminal epithelium by monocarboxylate transporter 1

Due to intensive intracellular metabolism of short-chain fatty acids, ruminal epithelial cells generate large amounts of D-beta-hydroxybutyric acid, acetoacetic acid, and lactic acid. These acids have to be extruded from the cytosol to avoid disturbances of intracellular pH (pH(i)). To evaluate acid extrusion, pH(i) was studied in cultured ruminal epithelial cells of sheep using the pH-sensitive fluorescent dye 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein. Extracellular addition of D-beta-hydroxybutyrate, acetoacetate, or lactate (20 mM) resulted in intracellular acidification. Vice versa, removing extracellular D-beta-hydroxybutyrate, acetoacetate, or lactate after preincubation with the respective monocarboxylate induced an increase of pH(i). Initial rate of pH(i) decrease as well as of pH(i) recovery was strongly inhibited by pCMBS (400 microM) and phloretin (20 microM). Both cultured cells and intact ruminal epithelium were tested for the possible presence of proton-linked monocarboxylate transporter (MCT) on both the mRNA and protein levels. With the use of RT-PCR, mRNA encoding for MCT1 isoform was demonstrated in cultured ruminal epithelial cells and the ruminal epithelium. Immunostaining with MCT1 antibodies intensively labeled cultured ruminal epithelial cells and cells located in the stratum basale of the ruminal epithelium. In conclusion, our data indicate that MCT1 is expressed in the stratum basale of the ruminal epithelium and may function as a main mechanism for removing ketone bodies and lactate together with H+ from the cytosol into the blood.     

Bovine rumen epithelium undergoes rapid structural adaptations during grain-induced subacute ruminal acidosis

Alterations in rumen epithelial structure and function during grain-induced subacute ruminal acidosis (SARA) are largely undescribed. In this study, four mature nonlactating dairy cattle were transitioned from a high-forage diet (HF; 0% grain) to a high-grain diet (HG; 65% grain). After feeding the HG diet for 3 wk, the cattle were transitioned back to the original HF diet, which was fed for an additional 3 wk. Continuous ruminal pH was measured on a weekly basis, and rumen papillae were biopsied during the baseline and at the first and final week of each diet. The mean, minimum, and maximum daily ruminal pH were depressed (P < 0.01) in the HG period compared with the HF period. During the HG period, SARA was diagnosed only during week 1, indicating ruminal adaptation to the HG diet. Microscopic examination of the papillae revealed a reduction (P < 0.01) in the stratum basale, spinosum, and granulosum layers, as well as total depth of the epithelium during the HG period. The highest (P < 0.05) papillae lesion scores were noted during week 1 when SARA occurred. Biopsied papillae exhibited a decline in cellular junctions, extensive sloughing of the stratum corneum, and the appearance of undifferentiated cells near the stratum corneum. Differential mRNA expression of candidate genes, including desmoglein 1 and IGF binding proteins 3, 5, and 6, was detected between diets using qRT-PCR. These results suggest that the structural integrity of the rumen epithelium is compromised during grain feeding and is associated with the differential expression of genes involved in epithelial growth and structure.     

Distributions of fibronectin, laminin, type I and IV collagens and carbonic anhydrase isozyme III during bovine ruminal epithelial development

The present study describes the immunohistochemical distributions of extracellular matrices and the carbonic anhydrase isozyme III (CA-3) in the bovine ruminal epithelium during fetal development. Fibronectin (FN), laminin and type I and IV collagens were distributed in the ruminal subepithelial mesenchymal regions with nonspecific regionality during the gestational periods. At stages after about 59 cm crown-rump length (CRL), FN of the epithelial basement membrane disappeared, and CA-3 appeared in the basal epithelial cells of the root of the ruminal papillae, suggesting that the functional differentiation of the ruminal epithelium might start at around 59 cm CRL in bovine fetuses.     

Effects of seasonal changes in food quality and food intake on the transport of sodium and butyrate across ruminal epithelium of reindeer

Transport of 22Na and 14C-butyrate across the ruminal epithelium of captive reindeer fed a concentrate diet in summer (n=5) and in winter (n=5) and from free-ranging reindeer taken from summer (n=3) and winter pasture (n=5) was measured in vitro in Ussing chambers. Significant amounts of both Na+ and butyrate were transported across the isolated epithelium without any external driving force. The ruminal transport of Na+ and butyrate were interacting, as evidenced by both the observed amiloride-induced reduction of net butyrate-transport and by the positive correlation between net transport of butyrate and Na+. Amiloride also reduced the net transport of Na+ without significantly affecting the short-circuit current, indicating the presence of an apical Na+/H+ exchanger in the ruminal epithelium of reindeer. The captive reindeer increased the dry matter intake of a constant quality concentrate from winter to summer, but this neither affected their ruminal transport capacity nor their ruminal surface enlargement factor (SEF). Free-ranging reindeer increased their ruminal transport capacity for Na+ and butyrate from summer to winter but simultaneously reduced their ruminal SEF. The present data indicate that this food-induced increase in transport capacity was attributed to changes in the nutrient composition of the diet.     

Stability of the glandular morphogenesis produced by retinoids in the newborn hamster cheek pouch in vitro

Retinoids can induce alterations in differentiation and morphogenesis in the hamster cheek pouch. In order to determine the stability of these changes, explants of neonatal pouch were exposed to 6 micrograms/ml of either retinyl acetate (RAc: 1.8 x 10(-5) M) or all-trans retinoic acid (RA: 2.0 x 10(-5) M) for an initial 3 of 7 days, out of a total of 21 days in organ culture. Three days of RAc or RA caused a delay in the differentiation and keratinization of the epithelium at least up to day 7 of culture. Additionally, two out of ten explants exposed to RA showed small downgrowths of epithelium into the stroma at 7 or 14 days. Seven days of exposure to either retinoid led to inhibition of epithelial keratinization, and produced a mucous metaplasia which was still seen at the end of the 21-day culture period. Periodic acid-Schiff (PAS)-positive, diastase-resistant material was present in the metaplastic epithelium, in intercellular, and in some instances, intracellular locations. An excess of either RAc or RA, for 7 days, induced persistent glandlike downgrowths of epithelium, suggesting that a stable alteration in the developmental program of the epithelium may have occurred. Many of these downgrowths possessed a lumen which was lined by cuboidal epithelium and contained PAS-positive, diastase-resistant secretory material. RA appeared more potent than RAc in inhibiting keratinization, in producing a mucous metaplasia, and in initiating glandlike downgrowths. The persistence of glandular downgrowths suggests that retinoids, either directly or indirectly, act in a manner similar to that of an embryonic inductor.     

The role of the mesenchyme in the development of the early primordium of the respiratory epithelium

Development of respiratory epithelium (RE) rudiment was studied in tissue culture after removal of mesenchyme (M) and respiratory tract (RT) in 13 days old embryos of A and C57BL mice. During long-term cultivation of intact RT, organotypic structures (branching bronchioles, alveolus-like cavities) developed. No epithelial organotypic structures developed in the presence of single M cells; explants were represented by layers of cubic epithelium. During long-term cultivation foci of atypical growth consisting of intensively proliferating basophilic cells with high nucleocytoplasmic ratio appeared in these explants. Regions of planocellular metaplasia with or without keratinization could be found in these foci. The frequency of atypical proliferates depended on the strain of donor mice and on the region of the explanted RT.     

Cytological response of palatal epithelium to TiN-coated CoCr alloy denture

The aim of this study was to assess the impact of titanium nitride coatings on CoCr alloy metal parts in framework dentures on human palatal epithelium cytology compared to framework dentures made with the same alloy but without titanium nitride coating, and to acrylic dentures. Every prosthetic restoration introduced into the oral cavity and remaining in direct contact with the palate exhibits a varied and harmful effect on the state of the palatal epithelium by disturbing its keratinization. CoCr alloy dentures produce a significantly greater perturbation of keratinization compared to acrylic dentures. There is no evidence showing that a titanium nitride coating of the CoCr alloy plays a protective role in the environment of the oral cavity.     

Involvement of activin signaling in abnormalities of mouse vagina exposed neonatally to diethylstilbestrol

Perinatal exposure to a synthetic estrogen, diethylstilbestrol (DES), causes cervicovaginal adenosis and permanent hyperplastic cornified vaginal epithelium with keratinization in mice. To investigate the mechanisms of the induction of vaginal abnormalities by DES, we have focused on activin A signaling. We have found that the βA-subunit mRNA is mainly expressed in the neonatal vaginal stroma, whereas activin A receptor type IB is localized in the neonatal vaginal epithelium. SMAD2, the intracellular signaling protein, is phosphorylated in the neonatal vagina. Cell proliferation in the vaginal epithelium grown in vitro is reduced by DES treatment or by activin signaling suppression through inhibin treatment. Thus, activin A (a homodimer of the βA-subunit) in the stroma stimulates epithelial cell proliferation in the neonatal vagina. DES treatment decreases the expression of the βA-subunit and activin receptor IIB but increases the expression of the βB-subunit and inhibin receptor. Neonatal DES treatment inhibits the phosphorylation of SMAD2 in the vaginal epithelium, indicating the inhibition of activin A signaling in the vaginal epithelium by neonatal DES treatment. Treatment with DES or inhibin, a native antagonist of activin, induces adenosis-like structures and keratinization in the vagina grown in vitro. These data suggest that the suppression of activin A signaling by DES is involved in the induction of cervicovaginal adenosis and keratinization in the neonatal mouse vaginal epithelium.     

EPIDERMAL METAPLASIA OF THE CORNEAL ANTERIOR EPITHELIUM IN THE CHICK EMBRYO

The corneal anterior epithelium of younger chick embryos can be changed into a keratinized epidermis, when it is cultured in vitro combined with 6 1/2-day dorsal dermis. Even if a Millipore filter is inserted between the corneal anterior epithelium and underlying dorsal dermis, the epithelium undergoes similar metaplastic changes. In older embryos, however, the epithelium gradually loses the competence for the keratinization. Cultivation of cornea (anterior epithelium, stroma and endothelium) of 6 1/2- or 10-day embryos results in maintenance of its original pattern, and the epithelium fails to differentiate into a keratinized epidermis. The dermis isolated from 8 1/2-day dorsal or 12 1/2-day tarsometatarsal skin is not so effective in inducing the epidermal metaplasia. The mesenchyme of 5 1/2-day proventriculus or 5 1/2-day gizzard fails to bring about any endodermal metaplasia of the corneal epithelium. The corneal stroma, on the other hand, has no inhibitory action on the keratinization of the epidermis obtained from 6 1/2-day dorsal skin.