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British Poultry Science Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/cbps20
Studies on the isthmus region of the domestic fowl Sarah E. Solomon
a
a
Department of Veterinary Histology and Embryology , University of Glasgow Veterinary School , Bearsden, Glasgow, Scotland Published online: 08 Nov 2007.
To cite this article: Sarah E. Solomon (1975) Studies on the isthmus region of the domestic fowl, British Poultry Science, 16:3, 255-258, DOI: 10.1080/00071667508416185 To link to this article: http://dx.doi.org/10.1080/00071667508416185
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http://www.tandfonline.com/page/termsand-conditions
Br. Poult. Sei., 16: 255-258.
1975
Longman:
printed in Great Britain
STUDIES ON THE ISTHMUS REGION OF THE DOMESTIC FOWL SARAH E. SOLOMON
Downloaded by [University of Calgary] at 17:47 01 February 2015
Department of Veterinary Histology and Embryology, University of Glasgow Veterinary School, Bearsden, Glasgow, Scotland Received for publication 27th March 1974
1. The isthmus extends from the aglandular zone, which delimits it from the magnum, to the tubular shell gland, which to the naked eye is marked by a distinct colour change from off-white to brown. 2. The surface epithelium comprises three cell types, ciliated, non-ciliated and mitochondrial, of which only the non-ciliated cells contribute towards the carbohydrate moiety of the shell membranes. 3. The gland cells are distinctive, containing granules of variable electron density, variations also occurring within individual granules. 4. Although two types of gland cell have been observed, they may merely represent different phases of development. 5. In the type 1 cell, the rough endoplasmic reticulum and Golgi complex are typical of the normal protein secreting cell; in the type 2 cell the RER is sparse, dilated and filled with intracisternal granules while the Golgi complex is likewise distended. INTRODUCTION
There is still some controversy about the extent and micro-structure of the isthmus, the region usually thought to produce the two soft shell membranes (Pearl and Curtis, 1914; Richardson, 1935). Thus it is considered by some (Leonard, 1968; Draper et al., 1972) to extend to the point at which the reproductive tract becomes expanded to form a pouch-like dilatation whereas Johnston et al. (1963) suggested that the lower limit of the isthmus was indicated by a colour change from the dull white characteristic of the latter to the brown colour characteristic of the shell gland. Ultrastructural analyses of this region have also been at variance. Johnston et al. (1963) described a transition zone between isthmus and shell gland characterised by the presence of complex secretory granules. Subsequently Khairallah (1966) reported granules of this type elsewhere in the isthmus. In this paper an attempt has been made to reconcile these various viewpoints and to relate them to the process of shell membrane formation. 255
256
SARAH E. SOLOMON
MATERIALS AND METHODS
Downloaded by [University of Calgary] at 17:47 01 February 2015
White Leghorn birds, approximately 1 year old were kept individually in wire cages and fed on a pelleted layer's diet and water. For ultrastructural investigation 30 birds were used, subdivided into 6 groups according to the position of the egg in the oviduct. Group 1 2 3 4 5 6
Position of egg Oviduct empty Upper magnum Lower magnum Isthmus (with membranes) Soft-membraned egg in shell gland Hard-shelled egg in shell gland
No. in group 3 4 5 6 5 7
The birds were anaesthetised with intravenous nembutal and the oviduct perfused via the aorta with 30 ml Karnovsky's fluid. The oviduct was removed, the position of the egg noted and small pieces of tissue 1 mm2 were removed from the areas depicted in Plate-Figs. 1-4. Following post-fixation in 1% buffered osmic acid, tissues were rinsed in distilled -water and dehydrated through an increasing series of concentrations of acetone before embedding in epon. Fine sections, 70 /um thick, were cut on an LKB Mark III Ultratome, mounted on uncoated copper grids and stained with uranyl acetate. For histochemical studies 14 birds were used, anaesthetised as described previously. The oviduct was opened, the position of the egg noted and pieces of material removed from the areas shown in Plate-Figs. 1-4. Tissues were fixed in 10% BNF overnight then dehydrated in an alcohol/chloroform series and embedded in paraffin wax. Sections were treated with periodic acid-Schiff (PAS), PAS/diastase and Biebrich Scarlet (Spicer and Lillie, 1961).
RESULTS
The distribution of cell types in the isthmus observed with the light microscope was in agreement with the findings of Draper et al. (1972). In the surface epithelium PAS-reactivity was confined to the non-ciliated cells (Plate-Fig. 2) and reactivity was diminished after the passage of an egg. The gland cells were PAS-positive in all parts of the isthmus and showed no variation in staining intensity with the phase of egg development. Treatment with diastase did not abolish the reaction, indicating the absence of glycogen. The isthmian granules stained strongly with Biebrich Scarlet at all stages of the laying cycle. At the ultrastructural level ciliated, non-ciliated and mitochondrial (PlateFigs 3 and 4) cells were identified, confirming the findings of Draper et al. (1972). The tubular glands presented a different appearance from that previously described and will be considered in more detail. The glands in cross-section comprise 4 or 5 pyramidal cells joined at their apices by desmosomes (Plate-Fig. 5). Two morphologically distinct types of cell were observed in the glands, in all parts of the isthmus, throughout the laying cycle and are hereafter referred to as type 1
ULTRASTRUCTURE OF THE ISTHMUS
257
and type 2 cells. Although no attempt was made to estimate the proportion of the two types, the type 2 cell was most conspicuous after the passage of the egg.
Downloaded by [University of Calgary] at 17:47 01 February 2015
Type 1 cell
Type 1 cell was characterised by the presence of numerous granules of variable size and electron density; many of the granules comprising two substances of differing electron density. The basally situated nucleus lay in association with the Golgi complex which surrounded smaller granules in various stages of development (Plate-Fig. 5). The rough endoplasmic reticulum (RER) varied in its location and degree of distension, sometimes appearing as parallel profiles in the lower two-thirds of the cell in close association with oval-shaped mitochondria. At other times it occurred throughout the cytoplasm in the form of dilated cisternae filled with a fibrillar type of material. Microvilli projected from the luminal surface of these cells. In some lumina the material was organised into an electron dense core surrounded by material of low electron density. The density of the secreted material was similar to the density of the intracellular granules and the latter appeared to fuse with the luminal cytosome and release their contents as amorphous masses. Type 2 cells
Type 2 cells also contained granules of variable density, although those of high electron density predominated. The Golgi complex which was supranuclear was invariably distended (Plate-Fig. 6). The most conspicuous feature of these cells was the RER which took the form of short cords scattered throughout the cytoplasm. Within the reticular cisternae were numerous discrete electron dense granules (Plate-Fig. 7). Frequently the tips of these cisternae were expanded to form smooth surfaced vesicles. Dilations, however, were also observed at points along the length of the cisternae. The material filling the gland lumina of these cells was of high electron density, similar to that of the granules filling the gland cells. DISCUSSION
The structure of the egg membrane fibres is reflected in the material filling some of the gland lumina. Initially, however, the secretory material is not discretely organised into core and mantle; how this reorganisation comes about is a matter of conjecture. It might be explained by a cross linking of adjacent functional groups on the protein molecules forming an irregular polymer in the centre, leaving the less dense carbohydrate sheath on the outside. The material filling the gland lumina originates from the numerous intracellular granules of variable electron density, which according to Khairallah (1966) are a complex of protein and carbohydrate. The present histochemical findings lend support to this view and also suggest that additional carbohydrate derives from the non-ciliated cells of the surface epithelium which contain less secretory material after the passage of the egg. This corroborates the findings of Robinson et al. (1968). Since these granules have been observed in all parts of the isthmus, it must be assumed that the entire region is involved in membrane formation. This concurs with the findings of Draper et al. (1972) and refutes the earlier suggestion of Leonard (1968) that the shell membranes are secreted specifically by the upper isthmus. 16/3—c
Downloaded by [University of Calgary] at 17:47 01 February 2015
258
SARAH E. SOLOMON
The results of Johnston et al. (1963) must also be modified in the light of these recent findings, since the granules which they found in the gland cells of the " isthmouterine junction " and which Simkiss (1968) implicated in the initiation of crystal formation occur in all parts of the isthmus. The variance in electron density possibly reflects different stages in the formation of one or, at most, two types of mature protein granule. The present findings are in agreement with those of Khairallah (1966) and Draper et al. (1972) with respect to the presence of intracisternal granules in the isthmian gland cells, however there is some disagreement about their temporal and spatial distribution. According to Khairallah ( 1966) these granules only occur when there is an egg in the isthmus, while Draper et al. (1972) suggested that they are a constant feature of all the gland cells. In this study intracisternal granules were observed in only a proportion of the gland cells but were found in all parts of the isthmus at all stages of the laying cycle. Kallenbach (1972) observed a similar accumulation of secretory material in rat preameloblasts and suggested that the intramembranous storage of protein precursor is the result of a lack of establishment between the RER and Golgi. This " block " in transport resulted in a marked vesiculation of the Golgi apparatus. Within the present system, it would appear that if such a breakdown is occurring, its initiation is independent of the state of secretory activity, since cells of this type invariably contain electron dense secretory granules. The mature membrane fibres do not appear to be released until local pressure is exerted by the descending ovum. The weak cohesion between the albumen and membranes possibly being a function of light end bonding between their respective acid and base groups. REFERENCES DRAPER, M. H., DAVIDSON, M. F., WYBURN, G. M. AND JOHNSTON, H. S. (1972). The fine structure
of the fibrous membrane forming region of the isthmus of the oviduct of Gallus domesticus. Q_. Jl exp. Physiol., 57: 297. JOHNSTON, H. S., ATTKEN, R. N. C. AND WYBURN, G. M. (1963). The fine structure of the uterus of
the domestic fowl. J. Anat., 97: 333. KALLENBACH, E. (1972). Granules in cisternae of the rough endoplasmic reticulum (RER) of preameloblasts and ameloblasts and a possible function of the RER in preameloblasts of rat incisor. J. Ultrastruct. Res., 39: 96. KHAIRALLAH, L. (1966). The Fine Structure of the Tubular Glands in the Isthmus of the Oviduct of the Hen. England, University microfilms. LEONARD, E. (1968). The accumulation of minerals in the avian oviduct. Ph.D. thesis, University of Edinburgh.
PEARL, R. AND CURTIS, M. R. (1912). Studies on the physiology of the domestic fowl. V : Data regarding the physiology of the oviduct. J. exp. Zool., 12: 99. RICHARDSON, K. C. (1935). The secretory phenomena in the oviduct of the fowl, including the process of shell formation examined by the microincineration technique. Phil. Trans. R. Soc., B225: 149. ROBINSON, D. S., KING, M. R. AND BOWEN, D. J . (1968). The occurrence of neutral and acidic mucins in the reproductive tract of the laying hen. Histochemie, 13: 97. SIMKISS, K. (1968). The structure and formation of the shell and shell membranes. In : Egg Quality: A study of the Hen's Egg, pp. 3-25 Edit. CARTER, T. C. Edinburgh, Oliver and Boyd. SPICER, S. S. AND LILLIE, F. C. (1961). Histochemical identification of basic proteins with Biebrich scarlet at alkaline p H . Stain Technol., 36: 365.
British Poultry Science Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/cbps20
Studies on the isthmus region of the domestic fowl Sarah E. Solomon
a
a
Department of Veterinary Histology and Embryology , University of Glasgow Veterinary School , Bearsden, Glasgow, Scotland Published online: 08 Nov 2007.
To cite this article: Sarah E. Solomon (1975) Studies on the isthmus region of the domestic fowl, British Poultry Science, 16:3, 255-258, DOI: 10.1080/00071667508416185 To link to this article: http://dx.doi.org/10.1080/00071667508416185
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http://www.tandfonline.com/page/termsand-conditions
Br. Poult. Sei., 16: 255-258.
1975
Longman:
printed in Great Britain
STUDIES ON THE ISTHMUS REGION OF THE DOMESTIC FOWL SARAH E. SOLOMON
Downloaded by [University of Calgary] at 17:47 01 February 2015
Department of Veterinary Histology and Embryology, University of Glasgow Veterinary School, Bearsden, Glasgow, Scotland Received for publication 27th March 1974
1. The isthmus extends from the aglandular zone, which delimits it from the magnum, to the tubular shell gland, which to the naked eye is marked by a distinct colour change from off-white to brown. 2. The surface epithelium comprises three cell types, ciliated, non-ciliated and mitochondrial, of which only the non-ciliated cells contribute towards the carbohydrate moiety of the shell membranes. 3. The gland cells are distinctive, containing granules of variable electron density, variations also occurring within individual granules. 4. Although two types of gland cell have been observed, they may merely represent different phases of development. 5. In the type 1 cell, the rough endoplasmic reticulum and Golgi complex are typical of the normal protein secreting cell; in the type 2 cell the RER is sparse, dilated and filled with intracisternal granules while the Golgi complex is likewise distended. INTRODUCTION
There is still some controversy about the extent and micro-structure of the isthmus, the region usually thought to produce the two soft shell membranes (Pearl and Curtis, 1914; Richardson, 1935). Thus it is considered by some (Leonard, 1968; Draper et al., 1972) to extend to the point at which the reproductive tract becomes expanded to form a pouch-like dilatation whereas Johnston et al. (1963) suggested that the lower limit of the isthmus was indicated by a colour change from the dull white characteristic of the latter to the brown colour characteristic of the shell gland. Ultrastructural analyses of this region have also been at variance. Johnston et al. (1963) described a transition zone between isthmus and shell gland characterised by the presence of complex secretory granules. Subsequently Khairallah (1966) reported granules of this type elsewhere in the isthmus. In this paper an attempt has been made to reconcile these various viewpoints and to relate them to the process of shell membrane formation. 255
256
SARAH E. SOLOMON
MATERIALS AND METHODS
Downloaded by [University of Calgary] at 17:47 01 February 2015
White Leghorn birds, approximately 1 year old were kept individually in wire cages and fed on a pelleted layer's diet and water. For ultrastructural investigation 30 birds were used, subdivided into 6 groups according to the position of the egg in the oviduct. Group 1 2 3 4 5 6
Position of egg Oviduct empty Upper magnum Lower magnum Isthmus (with membranes) Soft-membraned egg in shell gland Hard-shelled egg in shell gland
No. in group 3 4 5 6 5 7
The birds were anaesthetised with intravenous nembutal and the oviduct perfused via the aorta with 30 ml Karnovsky's fluid. The oviduct was removed, the position of the egg noted and small pieces of tissue 1 mm2 were removed from the areas depicted in Plate-Figs. 1-4. Following post-fixation in 1% buffered osmic acid, tissues were rinsed in distilled -water and dehydrated through an increasing series of concentrations of acetone before embedding in epon. Fine sections, 70 /um thick, were cut on an LKB Mark III Ultratome, mounted on uncoated copper grids and stained with uranyl acetate. For histochemical studies 14 birds were used, anaesthetised as described previously. The oviduct was opened, the position of the egg noted and pieces of material removed from the areas shown in Plate-Figs. 1-4. Tissues were fixed in 10% BNF overnight then dehydrated in an alcohol/chloroform series and embedded in paraffin wax. Sections were treated with periodic acid-Schiff (PAS), PAS/diastase and Biebrich Scarlet (Spicer and Lillie, 1961).
RESULTS
The distribution of cell types in the isthmus observed with the light microscope was in agreement with the findings of Draper et al. (1972). In the surface epithelium PAS-reactivity was confined to the non-ciliated cells (Plate-Fig. 2) and reactivity was diminished after the passage of an egg. The gland cells were PAS-positive in all parts of the isthmus and showed no variation in staining intensity with the phase of egg development. Treatment with diastase did not abolish the reaction, indicating the absence of glycogen. The isthmian granules stained strongly with Biebrich Scarlet at all stages of the laying cycle. At the ultrastructural level ciliated, non-ciliated and mitochondrial (PlateFigs 3 and 4) cells were identified, confirming the findings of Draper et al. (1972). The tubular glands presented a different appearance from that previously described and will be considered in more detail. The glands in cross-section comprise 4 or 5 pyramidal cells joined at their apices by desmosomes (Plate-Fig. 5). Two morphologically distinct types of cell were observed in the glands, in all parts of the isthmus, throughout the laying cycle and are hereafter referred to as type 1
ULTRASTRUCTURE OF THE ISTHMUS
257
and type 2 cells. Although no attempt was made to estimate the proportion of the two types, the type 2 cell was most conspicuous after the passage of the egg.
Downloaded by [University of Calgary] at 17:47 01 February 2015
Type 1 cell
Type 1 cell was characterised by the presence of numerous granules of variable size and electron density; many of the granules comprising two substances of differing electron density. The basally situated nucleus lay in association with the Golgi complex which surrounded smaller granules in various stages of development (Plate-Fig. 5). The rough endoplasmic reticulum (RER) varied in its location and degree of distension, sometimes appearing as parallel profiles in the lower two-thirds of the cell in close association with oval-shaped mitochondria. At other times it occurred throughout the cytoplasm in the form of dilated cisternae filled with a fibrillar type of material. Microvilli projected from the luminal surface of these cells. In some lumina the material was organised into an electron dense core surrounded by material of low electron density. The density of the secreted material was similar to the density of the intracellular granules and the latter appeared to fuse with the luminal cytosome and release their contents as amorphous masses. Type 2 cells
Type 2 cells also contained granules of variable density, although those of high electron density predominated. The Golgi complex which was supranuclear was invariably distended (Plate-Fig. 6). The most conspicuous feature of these cells was the RER which took the form of short cords scattered throughout the cytoplasm. Within the reticular cisternae were numerous discrete electron dense granules (Plate-Fig. 7). Frequently the tips of these cisternae were expanded to form smooth surfaced vesicles. Dilations, however, were also observed at points along the length of the cisternae. The material filling the gland lumina of these cells was of high electron density, similar to that of the granules filling the gland cells. DISCUSSION
The structure of the egg membrane fibres is reflected in the material filling some of the gland lumina. Initially, however, the secretory material is not discretely organised into core and mantle; how this reorganisation comes about is a matter of conjecture. It might be explained by a cross linking of adjacent functional groups on the protein molecules forming an irregular polymer in the centre, leaving the less dense carbohydrate sheath on the outside. The material filling the gland lumina originates from the numerous intracellular granules of variable electron density, which according to Khairallah (1966) are a complex of protein and carbohydrate. The present histochemical findings lend support to this view and also suggest that additional carbohydrate derives from the non-ciliated cells of the surface epithelium which contain less secretory material after the passage of the egg. This corroborates the findings of Robinson et al. (1968). Since these granules have been observed in all parts of the isthmus, it must be assumed that the entire region is involved in membrane formation. This concurs with the findings of Draper et al. (1972) and refutes the earlier suggestion of Leonard (1968) that the shell membranes are secreted specifically by the upper isthmus. 16/3—c
Downloaded by [University of Calgary] at 17:47 01 February 2015
258
SARAH E. SOLOMON
The results of Johnston et al. (1963) must also be modified in the light of these recent findings, since the granules which they found in the gland cells of the " isthmouterine junction " and which Simkiss (1968) implicated in the initiation of crystal formation occur in all parts of the isthmus. The variance in electron density possibly reflects different stages in the formation of one or, at most, two types of mature protein granule. The present findings are in agreement with those of Khairallah (1966) and Draper et al. (1972) with respect to the presence of intracisternal granules in the isthmian gland cells, however there is some disagreement about their temporal and spatial distribution. According to Khairallah ( 1966) these granules only occur when there is an egg in the isthmus, while Draper et al. (1972) suggested that they are a constant feature of all the gland cells. In this study intracisternal granules were observed in only a proportion of the gland cells but were found in all parts of the isthmus at all stages of the laying cycle. Kallenbach (1972) observed a similar accumulation of secretory material in rat preameloblasts and suggested that the intramembranous storage of protein precursor is the result of a lack of establishment between the RER and Golgi. This " block " in transport resulted in a marked vesiculation of the Golgi apparatus. Within the present system, it would appear that if such a breakdown is occurring, its initiation is independent of the state of secretory activity, since cells of this type invariably contain electron dense secretory granules. The mature membrane fibres do not appear to be released until local pressure is exerted by the descending ovum. The weak cohesion between the albumen and membranes possibly being a function of light end bonding between their respective acid and base groups. REFERENCES DRAPER, M. H., DAVIDSON, M. F., WYBURN, G. M. AND JOHNSTON, H. S. (1972). The fine structure
of the fibrous membrane forming region of the isthmus of the oviduct of Gallus domesticus. Q_. Jl exp. Physiol., 57: 297. JOHNSTON, H. S., ATTKEN, R. N. C. AND WYBURN, G. M. (1963). The fine structure of the uterus of
the domestic fowl. J. Anat., 97: 333. KALLENBACH, E. (1972). Granules in cisternae of the rough endoplasmic reticulum (RER) of preameloblasts and ameloblasts and a possible function of the RER in preameloblasts of rat incisor. J. Ultrastruct. Res., 39: 96. KHAIRALLAH, L. (1966). The Fine Structure of the Tubular Glands in the Isthmus of the Oviduct of the Hen. England, University microfilms. LEONARD, E. (1968). The accumulation of minerals in the avian oviduct. Ph.D. thesis, University of Edinburgh.
PEARL, R. AND CURTIS, M. R. (1912). Studies on the physiology of the domestic fowl. V : Data regarding the physiology of the oviduct. J. exp. Zool., 12: 99. RICHARDSON, K. C. (1935). The secretory phenomena in the oviduct of the fowl, including the process of shell formation examined by the microincineration technique. Phil. Trans. R. Soc., B225: 149. ROBINSON, D. S., KING, M. R. AND BOWEN, D. J . (1968). The occurrence of neutral and acidic mucins in the reproductive tract of the laying hen. Histochemie, 13: 97. SIMKISS, K. (1968). The structure and formation of the shell and shell membranes. In : Egg Quality: A study of the Hen's Egg, pp. 3-25 Edit. CARTER, T. C. Edinburgh, Oliver and Boyd. SPICER, S. S. AND LILLIE, F. C. (1961). Histochemical identification of basic proteins with Biebrich scarlet at alkaline p H . Stain Technol., 36: 365.
