Carbonic anhydrase generates CO2 and H+ that drive spider silk formation via opposite effects on the terminal domains.

Spider silk fibers are produced from soluble proteins (spidroins) under ambient conditions in a complex but poorly understood process. Spidroins are highly repetitive in sequence but capped by nonrepetitive N- and C-terminal domains (NT and CT) that are suggested to regulate fiber conversion in similar manners. By using ion selective microelectrodes we found that the pH gradient in the silk gland is much broader than previously known. Surprisingly, the terminal domains respond in opposite ways when pH is decreased from 7 to 5: Urea denaturation and temperature stability assays show that NT dimers get significantly stabilized and then lock the spidroins into multimers, whereas CT on the other hand is destabilized and unfolds into ThT-positive ß-sheet amyloid fibrils, which can trigger fiber formation. There is a high carbon dioxide pressure (pCO2) in distal parts of the gland, and a CO2 analogue interacts with buried regions in CT as determined by nuclear magnetic resonance (NMR) spectroscopy. Activity staining of histological sections and inhibition experiments reveal that the pH gradient is created by carbonic anhydrase. Carbonic anhydrase activity emerges in the same region of the gland as the opposite effects on NT and CT stability occur. These synchronous events suggest a novel CO2 and proton-dependent lock and trigger mechanism of spider silk formation.

Morphology and composition of the spider major ampullate gland and dragline silk.

Spider silk is made of unique proteins-spidroins-secreted and stored as a protein solution (dope) in specialized glands. The major ampullate gland, source of the dragline silk, is composed of a tail, a sac and an elongated duct. For this gland, several different types of epithelial cells and granules have been described, but it is largely unknown how they correlate with spidroin production. It is also not settled what parts of the large spidroins end up in the final silk, and it has been suggested that the N-terminal domain (NT) is lacking. Here we show that NT is present in the dope and throughout dragline silk fibers, including the skin layer, and that the major ampullate tail and sac consist of three different and sharply demarcated zones (A-C), each with a distinct epithelial cell type. Finally, we show that spidroins are produced in the A and B zone epithelia, while the C zone granules lack spidroins.

Age-related changes in the shell gland and duodenum in relation to shell quality and bone strength in commercial laying hen hybrids.

BACKGROUND: During the production period of laying hens, the number of cracked eggshells increases and the skeleton becomes brittle. Both these problems are related to ageing of the hen and cause economic problems for egg producers and impaired animal welfare. This study investigated key factors in the shell gland and duodenum related to eggshell quality and bone strength in laying hens during the production period. Five Lohmann Selected Leghorn (LSL) and five Lohmann Brown (LB), common hybrids in commercial egg production, were euthanized at 21, 29, 49 and 70 weeks (wk) of age. Blood samples for analysis of total calcium were taken at euthanization. Right femur and humerus were used for bone strength measurements and tissue samples from shell gland and duodenum were processed for morphology, immunohistochemical localisation of oestrogen receptors (ERα, ERß), plasma membrane calcium ATPase (PMCA) and histochemical localisation of carbonic anhydrases (CA). Eggs were collected for shell quality measurements. RESULTS: At age 49 week, shell and bone strength had both deteriorated, but the hens were then able to maintain the level until 70 week of age and femur bone strength even improved. The main physiological findings associated with the effects seen at 49 week were reduced gland density and a shift in balance between ERα and ERß in the shell gland, which coincided with a reduction in CA activity in the duodenum. Somewhat surprisingly, capillary density and capillaries with CA activity both increased in the shell gland over time, the latter possibly mediated via ERß. These findings were independent of hybrid. PMCA was found in both shell gland and duodenum, but appeared unrelated to the age-related changes in shell and bone quality. CONCLUSIONS: In hens around half-way through the production period, both shell quality and bone strength had deteriorated. Decreased gland density and a shift in the balance between ERα and ERß in the shell gland, co-occurring with a dramatic drop in duodenal CA activity, are suggested as possible factors involved in age-related changes in shell and bone quality.

Embryonic exposure to o,p'-DDT causes eggshell thinning and altered shell gland carbonic anhydrase expression in the domestic hen.

The mechanism for contaminant-induced eggshell thinning in wild birds remains to be clarified. It is generally assumed, however, that it results from exposure of the adult laying female. We have reported that embryonic exposure to the synthetic estrogen ethynylestradiol (EE2) results in eggshell thinning in the domestic hen. The objective of this study was to investigate whether eggshell thinning can be induced following in ovo exposure to a bioaccumulating estrogenic environmental contaminant, o,p'-DDT. Ethynylestradiol was used as a positive control. Domestic hens exposed in ovo to o,p'-DDT (37 or 75 microg/g egg) or EE2 (60 ng/g egg) laid eggs with thinner shells than the control birds. The hens from these exposure groups also had a significantly reduced frequency of shell gland capillaries with carbonic anhydrase (CA) activity, a key enzyme in eggshell formation. The decreased number of capillaries with CA activity suggests that a developmentally induced disruption of CA expression in the shell gland was involved in the eggshell thinning found in this study. Egg laying was not affected in hens exposed embryonically to 37 or 75 microg o,p'-DDT/g egg, whereas it was inhibited in hens exposed to higher doses. Decreased lengths of the left oviduct and its infundibulum were seen after embryonic treatment with o,p'-DDT or EE2. In addition, o,p'-DDT exposure resulted in right oviduct retention. The results support our hypothesis that eggshell thinning in avian wildlife can result from a functional malformation in the shell gland, induced by embryonic exposure to estrogenic substances.

Effects of intracerebroventricular infusion of genistein on gonadotrophin subunit mRNA and immunoreactivity of gonadotrophins and oestrogen receptor-alpha in the pituitary cells of the anoestrous ewe.

The present study was designed to demonstrate whether genistein, a synthetic phytoestrogen, infused into the third ventricle of the brain could affect the gonadotrophic cells regarding the presence of oestrogen receptor-alpha immunoreactivity and gonadotrophin subunit mRNA hybridising reaction in the ewe. Ewes (n=7), aged 2 years, in early anoestrous season were infused with Ringer-Locke solution (control, n=3) or 10 microg/100 microl/h of genistein (n=4) into the third ventricle over a 5 h period and slaughtered the following morning. Immunoreactivity of luteinizing hormone (LH), follicle-stimulating hormone (FSH) and oestrogen receptor-alpha (ERalpha) was determined in the adenohypophysis by immunohistochemistry using antibodies raised against LHbeta, FSHbeta, and ERalpha. Messenger RNA analyses were performed by non-isotope in situ hybridisation using sense and antisense riboprobes produced from beta subunits of LH and FSH cDNA clones. Computer image analysis was used to determine the percent of cells exhibiting immunohistochemical and/or hybridising reaction. It was found that in ewes infused with genistein, the percentage of LH-positive cells and the density of immunoreactive-LHbeta material decreased significantly (P

Carbonic anhydrase in mouse testis and epididymis; transfer of isozyme IV to spermatozoa during passage.

Spermatozoa are subjected to major changes as they pass through the epididymal duct. The aim of the present study was to describe the distribution of carbonic anhydrase (CA) in the mouse testis and epididymis using a histochemical technique showing total catalytic activity, in combination with immunohistochemistry for the two important isoforms CAs II and IV. By comparing normal mice with CA II-deficient mice, we were able to study membrane-bound CA without influence from the ubiquitous cytoplasmic CA II. Spermatozoa, when studied in both the scanning electron and light microscope, were found to pickup membrane-bound CA IV during their passage through the epididymal duct. The transfer appeared to take place in the proximal part of the corpus, where the apical membrane and vesicles of principal cells were richly supplied with CA IV. In addition to CA IV, another membrane-bound isozyme was located in basolateral membranes of principal cells. Cytoplasmic CA II was found in varying amounts in apical/narrow cells and principal cells of the corpus in control animals. The significance of CA for pH-regulating processes vital for sperm storage and motility is discussed. A function in HCO3- transport during sperm capacitation at fertilization is suggested for the CA IV found in spermatozoa.

Thyroid gland function in ovariectomized ewes exposed to phytoestrogens.

Phytoestrogens are by definition plant-derived substances that are able to activate the mammalian oestrogen receptors. We examined the possible effects of phytoestrogens on the secretion of thyroid hormones as well as on the immunoreactivity to oestrogen receptor alpha (ER alpha) in the thyroid glands of ovariectomized ewes. Eight ovariectomized ewes were fed 3.5 kg of 100% red clover silage for 14 days. Blood samples were collected before and on day 14 of exposure to phytoestrogens. After 5 months, four of the ewes were re-exposed to red clover silage as described above and the other four served as controls. Blood samples were collected as above. All ewes were slaughtered at the end of the experiment and the thyroid glands were weighed and examined for macroscopical changes. Tissue samples were taken for immunohistochemistry and image analysis. Ewes exposed to red clover silage had significantly higher plasma concentrations of total T(3) and free T(3) than ewes fed hay. The cross-section area of thyroid follicles tended to be larger in ewes fed red clover silage than in the control animals. ER alpha immunoreactivity was stronger in thyroid glands from ewes exposed to phytoestrogens than in ewes fed hay. In conclusion, daily ingestion of 81-95 mg phytoestrogens per kg body weight for 14 days stimulated secretion of thyroid hormones and tended to increase follicle size and ER alpha immunoreactivity of thyroid glands of ovariectomized ewes.

Exogenous estradiol improves shell strength in laying hens at the end of the laying period.

BACKGROUND: Cracked shells, due to age related reduction of shell quality, are a costly problem for the industry. Parallel to reduced shell quality the skeleton becomes brittle resulting in bone fractures. Calcium, a main prerequisite for both eggshell and bone, is regulated by estrogen in a complex manner. The effects of estrogen, given in a low continuous dose, were studied regarding factors involved in age related changes in shell quality and bone strength of laying hens. A pellet containing 0.385 mg estradiol 3-benzoate (21-day-release) or placebo was inserted subcutaneously in 20 birds each of Lohmann Selected Leghorn (LSL) and Lohmann Brown (LB) at 70 weeks of age. Eggs were collected before and during the experiment for shell quality measurements. Blood samples for analysis of total calcium were taken three days after the insertion and at sacrifice (72 weeks). Right femur was used for bone strength measurements and tissue samples from duodenum and shell gland were processed for morphology, immunohistochemical localization of estrogen receptors (ERα, ERß), plasma membrane calcium ATPase (PMCA) and histochemical localization of carbonic anhydrase (CA). RESULTS: Estrogen treatment increased shell thickness of both hybrids. In addition, shell weight and shell deformation improved in eggs from the brown hybrids. The more pronounced effect on eggs from the brown hybrid may be due to a change in sensitivity to estrogen, especially in surface epithelial cells of the shell gland, shown as an altered ratio between ERα and ERß. A regulatory effect of estrogen on CA activity, but not PMCA, was seen in both duodenum and shell gland, and a possible connection to shell quality is discussed. Bone strength was unaffected by treatment, but femur was stronger in LSL birds suggesting that the hybrids differ in calcium allocation between shell and bone at the end of the laying period. Plasma calcium concentrations and egg production were unaffected. CONCLUSIONS: A low continuous dose of estrogen improves shell strength but not bone strength in laying hens at the end of the laying period.

Effects of dietary phytoestrogens on plasma testosterone and triiodothyronine (T3) levels in male goat kids.

BACKGROUND: Exposure to xenoestrogens in humans and animals has gained increasing attention due to the effects of these compounds on reproduction. The present study was undertaken to investigate the influence of low-dose dietary phytoestrogen exposure, i.e. a mixture of genistein, daidzein, biochanin A and formononetin, on the establishment of testosterone production during puberty in male goat kids. METHODS: Goat kids at the age of 3 months received either a standard diet or a diet supplemented with phytoestrogens (3-4 mg/kg/day) for approximately 3 months. Plasma testosterone and total and free triiodothyronine (T3) concentrations were determined weekly. Testicular levels of testosterone and cAMP were measured at the end of the experiment. Repeated measurement analysis of variance using the MIXED procedure on the generated averages, according to the Statistical Analysis System program package (Release 6.12, 1996, SAS Institute Inc., Cary, NC, USA) was carried out. RESULTS: No significant difference in plasma testosterone concentration between the groups was detected during the first 7 weeks. However, at the age of 5 months (i.e. October 1, week 8) phytoestrogen-treated animals showed significantly higher testosterone concentrations than control animals (37.5 nmol/l vs 19.1 nmol/l). This elevation was preceded by a rise in plasma total T3 that occurred on September 17 (week 6). A slightly higher concentration of free T3 was detected in the phytoestrogen group at the same time point, but it was not until October 8 and 15 (week 9 and 10) that a significant difference was found between the groups. At the termination of the experiment, testicular cAMP levels were significantly lower in goats fed a phytoestrogen-supplemented diet. Phytoestrogen-fed animals also had lower plasma and testicular testosterone concentrations, but these differences were not statistically significant. CONCLUSION: Our findings suggest that phytoestrogens can stimulate testosterone synthesis during puberty in male goats by increasing the secretion of T3; a hormone known to stimulate Leydig cell steroidogenesis. It is possible that feedback signalling underlies the tendency towards decreased steroid production at the end of the experiment.

Development of sperm storage tubules in the quail during sexual maturation.

The development of the utero-vaginal sperm storage tubules (SST) in the quail during sexual maturation was studied using light microscopy and image analysis. SST development starts at around 28 days with low columnar cells, 10.9 +/- 0.7 microm high, found at the base of mucosal folds in the distal uterus. Seven days later, small bud-like invaginations consisting of columnar, 16.4 +/- 1.1 microm high cells with basal nuclei were found in this region. An extremely rapid growth of the oviduct occurred at approximately 40-42 days of age with considerable variation in oviductal length between birds, coefficient of variation (CV) 64.8% and 66.2%, respectively. Two of these birds had SST containing spermatozoa but were not laying. At 49 days, oviductal length was 24 +/- 0.5 cm (CV 2.0%), and all birds had functional SST with spermatozoa and had started to lay. Mature SST consist of columnar, nonciliated cells, 19.8 +/- 0.7 microm high. Although development of SST in the quail, to a large extent, coincides with the development of the rest of the oviduct, the present findings suggest that utero-vaginal sperm storage is possible before the complete maturation of the oviduct and subsequent onset of lay.

Reproductive impairment in Japanese quail (Coturnix japonica) after in ovo exposure to o,p'-DDT.

We have previously described various effects in adult Japanese quail consequent on treatment with oestrogenic compounds in ovo. In the present study, the environmental contaminant o,p'-DDT [1-(2-chlorophenyl)-1-(4-chlorophenyl)-2,2,2-trichloroethane] was administered to quail eggs to further evaluate test endpoints for oestrogenic effects related to reproduction in the Japanese quail. Exposure to 2 mg o,p'-DDT/egg (150 micro g/g egg) resulted in impaired sexual behaviour, reduced cloacal gland area and lowered plasma testosterone concentration in males. Females displayed oviductal abnormalities, including retained right oviduct, decreased length of left oviduct, alterations in shell gland morphology and disrupted distribution of carbonic anhydrase in the shell gland. Egg laying was severely impaired. Consequently, a number of endpoints in adult quail may be useful for demonstrating an oestrogen-like mode of action by environmental contaminants during embryonic development.