Effects of phytoestrogen supplementation in the feed on the shell gland of laying hens at the end of the laying period.

Shell quality decreases as laying hens age and the aim of present study was to investigate how a supplement of daidzein, a natural phytoestrogen in soya, affects key factors in the shell gland and eggshell quality in late-stage laying hens. Hybrids of Lohmann Selected Leghorn (LSL) and Lohmann Brown (LB), received either a daidzein diet (50 mg/kg feed) or a control diet from 60 to 72 weeks of age. Both the total number of capillaries and capillaries with carbonic anhydrase (CA) activity were higher in the LSL hybrid than in the LB. After daidzein supplementation the number of CA positive capillaries was unaffected in the LSL but increased in the LB hybrid indicating a higher sensitivity to daidzein in this hybrid. Estrogen receptor alpha and beta (ERα, ERß) were localized and the complete picture of the two ERs can now be described in shell gland of domestic hens. Nuclear and cytoplasmic staining was generally stronger for ERß, while membrane associated staining was present only for ERα. Interestingly, capillary endothelium contained only ERß and since estrogen regulation of CA is well documented, the presence of an endothelial ER provides one possible route for the increase in CA positive capillaries found in LB hybrids. Eggshell quality or egg production was not affected by daidzein supplementation. The hybrids used in this study showed anatomical differences and reacted differently to daidzein supplementation, but if this can be explained by the divergences in ERß localization noted between the hybrids remains to be clarified.

Defocused CO2 laser on equine skin: a histological examination.

REASONS FOR PERFORMING STUDY: No studies have been published on effects of treatment with a defocused beam carbon dioxide (CO2) laser on equine skin histology. A better understanding of this will help to define how lasers should be used, in order to reduce potential side effects. OBJECTIVE: To describe the acute effects of different doses of defocused CO2 laser, ranging from therapeutic to surgical levels, on equine skin. METHODS: Defocused CO2 laser was administered to the skin in the hamstrings (91 J/cm2), fetlock (137 J/cm2) and loin (450 J/cm2) areas of 13 Standardbred horses. The acute effects on skin histology were examined 90 min after the end of therapy. RESULTS: Mild changes with focal spongiosis and subepidermal clefts were found after 91 J/cm2 irradiation and more severe changes with diffuse subepidermal clefts after the 137 J/cm2 dose. A homogeneous eosinophilic acellular zone of dermis and destruction of adnexal structures, and significant thinning of the epidermis was observed after the 450 J/cm2 dose. CONCLUSIONS: The present study indicates acute dose-dependent changes in equine skin histology after laser treatment Severe tissue damage was induced using a 450 J/cm2 dose. POTENTIAL RELEVANCE: To reduce the potential side effects of defocused CO2 laser treatment, the laser parameters must be carefully evaluated. Caution should be taken if doses higher than 91 J/cm2 (16 W, 4 min, and 42 cm2) are used in irradiation of equine skin.

Defective reproductive organ morphology and function in domestic rooster embryonically exposed to o,p'-DDT or ethynylestradiol.

Environmental pollutants with estrogenic activity have a potential to disrupt estrogen-dependent developmental processes. The objective of this study was to investigate if embryonic exposure to the environmental estrogens o,p'-DDT (1-(2-chlorophenyl)-1-(4-chlorophenyl)-2,2,2-trichloroethane; 37, 75, 150 or 300 microg/g egg) and EE2 (17alpha-ethynyl estradiol; 60 ng/g egg) affects the reproductive system in domestic roosters. Following egg injection on Embryonic Day 4, the newly hatched chicks were sexed by cloacal inspection. A skewed phenotypic sex ratio with overrepresentation of chicks deemed as females was observed in the groups exposed to the three highest doses of o,p'-DDT but not in the EE2-exposed group. Normal sex ratios were observed in all groups at adulthood. However, a cloacal deformation seemed to remain in the adult roosters, causing an abnormal semen flow upon semen collection. Semen yield was significantly reduced in both o,p'-DDT-exposed and EE2- exposed birds, whereas semen quality was unaffected. When killed, deformations of the left testis were found in all treatment groups. Image analysis revealed a reduced seminiferous tubular area in the roosters exposed to the two highest doses of o,p'-DDT. Embryonic exposure to o,p'-DDT caused decreased comb weight and right-spur diameter, while EE2 only affected right-spur diameter. In conclusion, this study shows that embryonic exposure to estrogenic compounds can induce permanent effects in male birds. The effects of the two studied compounds were partly similar but o,p'-DDT also induced alterations not seen in the EE2-treated birds.

Embryonic exposure to oestrogen causes eggshell thinning and altered shell gland carbonic anhydrase expression in the domestic hen.

Eggshell thinning among wild birds has been an environmental concern for almost half a century. Although the mechanisms for contaminant-induced eggshell thinning are not fully understood, it is generally conceived to originate from exposure of the laying adult female. Here we show that eggshell thinning in the domestic hen is induced by embryonic exposure to the synthetic oestrogen ethynyloestradiol. Previously we reported that exposure of quail embryos to ethynyloestradiol caused histological changes and disrupted localization of carbonic anhydrase in the shell gland in the adult birds, implying a functional disturbance in the shell gland. The objective of this study was to examine whether in ovo exposure to ethynyloestradiol can affect eggshell formation and quality in the domestic hen. When examined at 32 weeks of age, hens exposed to ethynyloestradiol in ovo (20 ng/g egg) produced eggs with thinner eggshells and reduced strength (measured as resistance to deformation) compared with the controls. These changes remained 14 weeks later, confirming a persistent lesion. Ethynyloestradiol also caused a decrease in the number of shell gland capillaries and in the frequency of shell gland capillaries with carbonic anhydrase activity. These data suggested that a disrupted carbonic anhydrase expression was involved in the mechanism for the oestrogen-induced eggshell thinning found in this study. The results support our hypothesis that eggshell thinning in avian wildlife can result from a structural and functional malformation in the shell gland, induced by xeno-oestrogen exposure during embryonic development.

Carbonic anhydrase activity and sweat gland morphology in trained and untrained Standardbred trotters.

Sweat gland morphology and carbonic anhydrase (CA) distribution was studied after exercise in trained and untrained horses using a histochemical technique and light microscopic image analysis. Three trained and 3 untrained Standardbred trotters performed an exercise test (20 min trot at 6 m/s with 5 min walk at 1.8 m/s in the beginning and end) on a high-speed treadmill at 35 degrees C. Skin biopsies were taken before exercise and after trot. The fluid loss after exercise was 10, 12 and 12 g/kg bwt in the untrained horses and 4, 6 and 11 g/kg in the trained. Trained horses had a larger cell area than untrained after exercise, which might be related to an increase in secretory capacity. The area of the cell occupied by CA was independent of training status, but increased with exercise in both groups. The CA activity was higher in untrained animals and increased after exercise in both groups. The change in CA during exercise might be a response to an increasing demand for HCO3- secretion during sweat formation. Therefore, the sweat gland undergoes morphological changes due to stimuli such as heat, exercise and training, but species differences are evident. To our knowledge, no one has previously studied the influence of training on the morphology of the equine sweat gland.

Localization of a protein inhibitor of carbonic anhydrase in pig tissues.

The protein inhibitor of carbonic anhydrase (CA), pICA, was localized in pig tissues by an immunohistochemical technique, using rabbit antipICA IgG. Staining for pICA was found in liver sinusoids and kidney glomeruli, where phagocytic cells are located, i.e. Kupffer and mesangial cells, respectively. pICA was not found inside parenchymal cells, or in tissues from striated muscle, heart, eye or lung. It is concluded that the function of pICA is perhaps to bind the carbonic anhydrase isozymes CA I, II, and III, released from erythrocytes into the blood circulation by intravascular haemolysis. The complex of CA-pICA in plasma may then be transported to the reticuloendothelial system, for degradation and reclamation of CA-bound zinc. This would be similar to the fate of the haemoglobin-haptoglobin complex for the recycling of iron.

Disrupted carbonic anhydrase distribution in the avian shell gland following in ovo exposure to estrogen.

Eggshell thinning among wild birds has been an environmental concern for almost half a century and the underlying mechanisms are still not fully understood. Previously we showed that exposure of quail embryos to ethynylestradiol (EE2) caused disorganization of the tubular glands in the shell gland of adult birds. In this study, we have examined the effect of in ovo exposure to EE2 on carbonic anhydrase (CA) localization, especially in the shell gland, because CA is required for shell formation. In the control birds, CA was localized in the cell membranes of the tubular gland cells of the shell gland, whereas the surface epithelium was always devoid of CA. In ovo treatment with 20ng EE2/g egg resulted in a loss of CA activity in the tubular glands while the surface epithelium showed strong induction of both membrane bound and cytoplasmic CA activity in 49+/-1% of the cells. The dose 2ng EE2/g egg resulted in partial loss of tubular gland CA and strong induction of CA activity in 2.5+/-0.5% of the surface epithelial cells and weaker induction in 22+/-2% of the epithelial cells. In conclusion, this study shows that embryonic exposure to a xenoestrogen disrupts CA distribution in the adult shell gland. We propose that eggshell thinning in avian wildlife could reflect a functional malformation in the shell gland, already induced by xenoestrogen during embryonic development rather than being caused solely by exposure of the adult bird.

Biochemical, histological, and inhibitor studies of membrane carbonic anhydrase in frog gastric acid secretion.

Gastric acid secretion is dependent on carbonic anhydrase (CA). To define the role of membrane-bound CA, we used biochemical, histochemical, and pharmacological approaches in the frog (Rana pipiens). CA activity and inhibition by membrane-permeant and -impermeant agents were studied in stomach homogenates and microsomal fractions. H(+) secretion in the histamine-stimulated isolated mucosa was measured before and after mucosal addition of a permeant CA inhibitor (methazolamide) and before and after mucosal or serosal addition of two impermeant CA inhibitors of differing molecular mass: a 3,500-kDa polymer linked to aminobenzolamide and p-fluorobenzyl-aminobenzolamide (molecular mass, 454 kDa). Total CA activity of frog gastric mucosa is 2,280 U/g, of which 10% is due to membrane-bound CA. Membrane-bound CA retains detectable activity below pH 4. Histochemically, there is membrane-associated CA in surface epithelial, oxynticopeptic, and capillary endothelial cells. Methazolamide reduced H(+) secretion by 100%, whereas the two impermeant inhibitors equally blocked secretion by 40% when applied to the mucosal side and by 55% when applied to the serosal side. The presence of membrane-bound CA in frog oxynticopeptic cells and its relative resistance to acid inactivation and inhibition by impermeant inhibitors demonstrate that it subserves acid secretion at both the apical and basolateral sides.

Carbonic anhydrase in the utero-vaginal junction of immature and mature ostriches.

1. Sperm storage tubules in the ostrich start to develop at an early stage of oviductal growth. Concurrently, membrane-bound carbonic anhydrase was found in the cells of the storage tubules. 2. In mature ostriches the utero-vaginal junction averaged 11.5+/-2.1 cm in length and primary mucosal folds were extremely long and slender. Membrane-bound carbonic anhydrase was present in the cells of the sperm storage tubules. In the non-ciliated cells of the surface epithelium both membrane-bound and cytoplasmic activity was detected. 3. The possible role of carbonic anhydrase in the stimulation/inhibition of sperm motility by altering the pH was discussed.

Localization of calcium and zinc in the sperm storage tubules of chicken, quail and turkey using X-ray microanalysis.

Sperm storage tubules from the utero-vaginal junction of chickens, quails and turkeys were analysed for calcium and zinc using X-ray microanalysis of ultra-rapidly frozen tissue in a scanning electron microscope. This technique enabled the tubular fluid surrounding the stored spermatozoa and the intracellular content of the cells of the sperm storage tubules to be analysed separately and, by using standards with known concentrations, their elemental concentrations were estimated. The mean (+/- SEM) concentration of calcium in the tubular fluid from chickens, quails and turkeys was 17 +/- 3, 19 +/- 3 and 17 +/- 4 mmol kg(-1) wet weight, respectively. The intracellular calcium concentration of the cells of the tubules did not differ significantly from these values and was also similar in the mucosal epithelial cells of the utero-vaginal junction. Zinc was localized in the cells of turkey sperm storage tubules and tubular fluid, but at low concentrations. No zinc could be detected in corresponding structures from chickens and quails. The concentration of calcium in the tubular fluid is within the range known to inhibit the motility of spermatozoa, supporting this function for calcium during storage. Zinc is known to depress turkey sperm metabolism and it may also be involved in inducing quiescence of spermatozoa during storage in this species.

Membrane-associated carbonic anhydrase activity in the brain of CA II-deficient mice.

Membrane-associated carbonic anhydrase (CA) activity is of importance for transepithelial transport of ions and fluid. Histochemical studies have indicated its presence in the brain, but the data are difficult to evaluate because of interference from cytoplasmic CA isozymes, of which CA II is the predominant one. CA II-deficient mice offer a possibility to study the location of membrane-associated CA-activity, without interference from CA II. The location of CA activity in the brain of CA II-deficient and normal mice was studied by the cobalt-phosphate histochemical method, and that of CA I, CA II and CA III by an immunocytochemical method. The brains of both types of mice lacked cytoplasmic isozymes CA I and CA III, and the CA II-deficient mice also lacked CA II. In the normal mice, oligodendrocytes and choroid epithelium stained for CA II in the cytoplasm. In normal and CA (II)D-mice there was an intense membrane associated histochemical CA activity in neuronal processes. Neuronal perikarya were not stained. Endothelial membranes of brain capillaries showed strong histochemical CA-activity. Choroid epithelial cells had histochemical CA activity in the cytoplasm and along apical and baso-lateral cell membranes. The results suggest that membrane-associated CA-activity found along neuronal processes probably modulates pH of the extracellular fluid and thus neuronal activity. CA II and the membrane-associated CA of choroidal epithelium are probably involved in the secretion of cerebrospinal fluid.

Sweat production and localisation of carbonic anhydrase in the equine sweat gland during exercise at two ambient temperatures.

The aim of this investigation was to study sweat production during exercise at 2 ambient temperatures (20 degrees C and 35 degrees C) and the concurrent localisation of carbonic anhydrase (CA) in the sweat gland. Horses develop alkalosis during prolonged exercise and the sweat contains HCO3-. Carbonic anhydrase is therefore of interest since it catalyses the reaction CO2 + H2O<-->HCO3- + H+. Four standardbred trotters performed an exercise test. Skin biopsies were taken from the neck, and sweat rate, blood and skin temperatures were measured. There was a close relationship between sweat rate, temperatures and work intensity at 20 degrees C. Temperatures and sweat rate were higher at 35 degrees C and did not fall when the work intensity dropped. A significant decrease in the sweat gland cell area was found after exercise at 35 degrees C with an accompanying decrease of vesicles. Strong CA activity was present at the luminal cell membrane and weaker basolaterally. The staining intensity increased after exercise. We suggest that CA might be of importance for counteracting the alkalosis developed after exercise by delivering HCO3- for generation of the alkaline pH in sweat.

Functional receptors for atrial natriuretic peptide in the rat mammary gland during lactation.

The present study was undertaken: 1) to localize and characterize atrial natriuretic peptide (ANP) receptors in the rat mammary gland; and 2) to elucidate ANP-induced cellular formation of cyclic GMP (cGMP) and alterations in alveolar morphology during both early and late lactation. Receptor autoradiography, employing rat-specific [125I]ANP as radioligand, demonstrated binding sites in the secretory tissue and larger blood vessels of the mammary gland. Binding of [125I]rANP to membrane fractions was completely displaced by unlabeled ANP and brain natriuretic peptide. C-type natriuretic peptide and cANP(4-23) revealed limited competition with radiolabeled ANP only during early lactation, indicating a more heterogeneous receptor population at that time. Systemically administered ANP induced cGMP formation in the alveolar epithelium, as shown with immunohistochemistry, and increased mammary tissue cGMP concentrations in vivo throughout the lactation period. Image analysis revealed enlargement of alveolar (but not epithelial) cell area after ANP stimulation in late lactation, suggesting altered alveolar filling or myoepithelial cell relaxation. These results indicate that ANP induces biological effects in the rat mammary gland through specific ANP-A receptor interaction with subsequent intracellular cGMP formation. ANP may therefore play a regulatory role in the control of mammary gland blood supply and secretory function.

Localization of carbonic anhydrase in the goat mammary gland during involution and lactogenesis.

The aim of this study was to determine whether carbonic anhydrase (CA) activity in goat mammary capillaries is regulated mainly by local or systemic mechanisms. One gland was dried before the contralateral gland, and after parturition only one gland was milked. Biopsies were taken from the mammary glands of three goats at 14 d intervals during involution and the start of the following lactation. A histochemical method was used to visualize sites of CA activity. To follow the involution process, milk (liquid) samples were taken from both teats each week and analysed for pH and composition. The time course of CA activity disappearance and reappearance in the capillaries was related to changes in milk composition and alveolar area. A dense network of capillaries showing membrane-bound staining for CA was found surrounding the alveoli in the lactating gland. CA activity gradually decreased in the drying gland, although the other gland was being milked. After 8 weeks involution the dried gland had a significantly lower number of stained capillaries than the milked gland. Almost no stained capillaries were found during late pregnancy, when both glands were dried and the tissue growth maximal. During lactation milk pH was 6.6 +/- 0.3 and this increased to 7.0 +/- 0.1 in the course of involution. In the last trimester of pregnancy the pH returned to its lower value, while the mammary gland was devoid of stained capillaries. Therefore, the capillary CA could not have been directly involved in the pH regulation of milk. The CA activity reappeared in the capillaries directly after delivery, but only in the milked gland. Clearly the regulation of CA activity is influenced more by local than by systemic factors and is associated with the metabolic activity of milk secretion.

Localization of carbonic anhydrase in the sperm-storing regions of the turkey and quail oviduct.

The localization of carbonic anhydrase in the sperm storage regions of turkey and quail was investigated using a histochemical method showing the activity of all the isozymes present. Intense carbonic anhydrase activity was found in the turkey sperm storage tubules and infundibular storage glands, whereas no activity could be detected in the quail at these sites. Both species did, however, show strong membrane-bound and cytoplasmic activity in the non-ciliated cells of the utero-vaginal surface epithelium and scattered cells of the vaginal epithelium. The enzyme catalyses the reaction CO2 + H2O <--> H+ + HCO3-, and the presence of carbonic anhydrase in these regions makes rapid changes in pH possible. It is suggested that increasing pH and/or the addition of bicarbonate stimulates sperm motility needed during transfer of the oviducal lumen. A lowering of the pH would keep the sperm quiescent during storage. The duration of sperm storage is considerably longer in the turkey than in the quail. The high quantity of carbonic anhydrase in the turkey sperm storage tubules may, thus, play a role in the duration of sperm storage.

Carbonic anhydrase activity in different placenta types: a comparative study of pig, horse, cow, mink, rat, and human.

The placenta has multiple functions, being the organ which provides oxygen and nutrients to the developing conceptus. In the placenta, the enzyme carbonic anhydrase (CA) may provide ions for exchange with Na+, K+, and Cl- in transepithelial movement of ions and fluid, as well as facilitating carbon dioxide diffusion. It can also be active in intermediary metabolism, such as gluconeogenesis, urea, and fatty acid synthesis. Placental material from pig, horse, cow, mink, rat, and human was therefore investigated, representing placenta types with variations in shape, internal architecture, and nature of the interhemal barrier. After glutaraldehyde fixation, sections were stained by a histochemical CA-method demonstrating all active isozymes. The most striking feature in common was a positive reaction in the maternal capillaries, when present, as in pig, horse, cow, and mink. In the maternal epithelium, the activation of CA was only observed in the pig, which also exhibited the strongest activity at the maternal interface, which reacted moderately in rat, weakly in horse, and was not visible in cow and human. The trophoblast was positive in pig and rat, whereas it was negative in horse, cow, human, and mink placentae except for few scattered trophoblast cells in pig, horse, and cow, which showed very intense activity. In the fetal capillaries, a positive reactivity was only observed in mink and human. The utilization of CA in placental transfer and metabolism is thus highest in the pig, rat, and mink, compared with horse, cow, and human. It can therefore be concluded that the activation and localization of CA in the placental interhemal barrier varies considerably among species.

Carbonic anhydrase and cardiac pH regulation.

Membrane-bound carbonic anhydrase (CA) has recently been identified in mammalian cardiac tissue. In this study, we have investigated the histochemical location and functional role of CA in the ferret heart. Heart sections stained by a modified Hansson's technique showed CA to be located on capillary endothelial membranes as well as on sarcolemmal membranes. In the Langendorff-perfused heart, washout of CO2 brought about by switching perfusion between 25 mM HCO3(-)-5% CO2-buffered solution and nominally HCO3(-)-CO2-free solution caused a transient rise in intracellular pH (pHi) measured by the chemical shift of 2-deoxy-D-glucose 6-phosphate with 31P nuclear magnetic resonance spectroscopy. The initial rate of change of pHi, measured over the first 60-75 s of CO2 efflux, was significantly reduced from 0.41 +/- 0.03 pH units/min (n = 9) in control hearts to 0.28 +/- 0.02 pH units/min (n = 5) in the presence of the membrane-permeable CA inhibitor 6-ethoxzolamide (P < 0.05 compared with control) and to 0.22 +/- 0.04 pH units/min (n = 5) in the presence of the membrane-impermeable CA inhibitor CL-11,366 (P < 0.01 compared with control). After reperfusion of the ischemic myocardium, both CA inhibitors caused a significant slowing of initial rate of change in pH (and initial rate of recovery of contractile function) compared with control hearts. These results suggest that CA, by facilitating the hydration-dehydration of CO2-H2CO3, alters the relative concentrations of CO2 inside and outside the cells, thus enhancing the rate of CO2 transfer from the intracellular to extracellular compartments, which contributes significantly to pHi recovery after reperfusion of the ischemic myocardium.

Localisation of carbonic anhydrase in the sperm storing regions of the domestic hen oviduct.

In the female bird sperm is stored in a quiescent mode, but full motility is needed for successful fertilisation. Regulation of sperm motility is thus of vital interest and the pH is a factor of importance. For this reason the localisation of carbonic anhydrase in the vagina, uterovaginal junction and infundibulum was studied with a histochemical method. Carbonic anhydrase catalyses the reaction CO2 + H2O <--> H+ + HCO3- and is known to take significant part in acid-base regulation in the body. The enzyme was found in all regions with the highest activity, both cytoplasmic and membrane-bound, in the non-ciliated cells of the uterovaginal surface epithelium. Intense membrane-bound activity was also found in the infundibular grooves and glands with slightly less in the sperm storage tubules and vaginal epithelium. Occasionally cytoplasmic and nuclear staining was seen. Changes in pH affect sperm motility and our results provide the first evidence for cellular mechanisms that makes rapid changes of the pH possible in these regions. Judging from the distribution of carbonic anhydrase we suggest two possible functions: (1) increasing pH and/or adding bicarbonate ions to stimulate sperm motility needed for the transfer to the storage sites and at fertilisation, and (2) a lowering of the pH in the sperm storage sites to keep the sperm quiescent during storage.

Membrane-associated CA activity in the eye of the CA II-deficient mouse.

PURPOSE: Membrane-associated carbonic anhydrase (CA) activity is probably of great importance for transepithelial transport of ions and fluid. Histochemical studies have indicated its presence in the eye, but such histochemical data are difficult to evaluate because of interference from cytoplasmic CA isozymes, of which CA II is predominant. CA II-deficient mice offered the possibility to study the localization of membrane-associated CA activity, without influence from CA II: METHODS: The localization of CA in the eyes of CA II-deficient mice and of normal mice was studied by the cobalt-phosphate histochemical method. RESULTS: In both types of mice, intense histochemical CA activity was associated with the apical and basolateral membranes of the pigmented and nonpigmented ciliary epithelium, of the corneal endothelium, and of the pigmented epithelium of the retina. It also was localized at the cell borders of the Müller cells and of the lens epithelium and fibers. There also was CA activity in the endothelium of the capillaries of the choroid and retina but not in that of the larger vessels. CONCLUSIONS: Membrane-associated CA activity is found in many ocular cells known to transport fluid and ions. Inhibition of the CA activity of the basolateral membranes of the ciliary nonpigmented epithelium probably explains the reduction of aqueous humor flow seen after the administration of CA inhibitors.