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A. Barua and Y. Yoshimura

The aim of this study was to localize major histocompatibility complex class II positive (MHC-II+) cells in the hen ovary, and to determine the effects of ageing and sex steroids on their frequency. Cryostat sections of ovarian tissues of immature, young laying and old laying hens and those of immature hens treated with or without diethylstilboestrol or progesterone were prepared. Sections were immunostained for MHC class II antigens using mouse anti-chicken MHC class II monoclonal antibody and observed under a light microscope. Positive cells were counted using a computer-assisted image analyser. MHC-II+ cells were localized in the ovarian stroma and theca layer of primary follicles in all birds examined. The frequency of MHC-II+ cells in the stroma and theca of primary follicles (approximately 400–600 μm in diameter) was significantly greater in young laying hens than it was in immature and old laying hens (P < 0.01). In the stroma and the theca of primary follicles of diethylstilboestrol-treated birds, the frequency of MHC-II+ cells was significantly greater than it was in the stroma and theca of control and progesterone-treated birds (P < 0.01). Progesterone had no significant effect when compared with controls. These results indicate that both the ovarian stroma and theca of follicles in the hen ovary contain MHC-II+ cells, the frequency of MHC-II+ cells increases in association with sexual maturation and decreases thereafter during ageing, and oestrogen may be one of the factors enhancing the induction of MHC-II+ cells in the ovary.

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A Barua and Y Yoshimura

The aim of this study was to determine whether anti-ovarian autoantibodies appear in the circulation of laying hens and whether the concentrations of these antibodies change with respect to ageing and egg laying rate. Autoantibodies to ovarian tissues in the circulation of aged (aged approximately 670 days) White Leghorn hens with low (< 50%) and high (> 90%) egg laying rates were examined by ELISA and western blotting. Young laying hens (aged 185 days) with > 95% egg production were used as controls. The results of the ELISA indicated that IgG, which bound to the ovary and small white follicles, was present in the circulation of old laying hens. More hens that laid few eggs had circulatory autoantibodies to the ovary and small white follicles, as determined by the cut-off value in ELISA (mean absorbance + 2 SD of young laying hens), than did hens that laid greater numbers of eggs, and the concentration of IgG was significantly higher in the hens that laid few eggs. In contrast, when the muscle proteins were used as antigens there were no significant differences in the absorbance values among low and high laying frequency old hens or young hens. Western blotting revealed many bands of immunoprecipitates formed by ovarian antigens and antibodies in the serum of old hens, indicating the presence of many binding sites for circulatory IgG in ovarian tissues. These results indicate that antibodies to ovarian tissues appear in the circulation of laying hens during ageing, and that the concentration of these autoantibodies is related inversely to the rate of egg laying by hens.

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Y. Yoshimura and J. M. Bahr

The germinal disc of the female gamete and its overlying follicular wall (the germinal disc region) is hypothesized to play an important role in the regulation of follicular growth. The role of the germinal disc region in follicular growth in chickens was investigated by destroying the germinal disc region or an area opposite the germinal disc (control) of the second largest follicle 22–23 h before ovulation of the F1 follicle, by localized freezing with solid CO2. Structural changes of the follicular wall (non-frozen region) were observed by electron microscopy 10–20 h after the destruction of the germinal disc region. Development of the inner structure of mitochondria in granulosa cells and accumulation of lipid droplets in thecal cells were observed in follicles 15 h after destruction of the germinal disc region. Twenty hours after destruction of the germinal disc region, follicles showed early signs of atresia (bursting atresia). Degenerative changes in follicles, including hydrolysis by lysosomal enzymes, were present in thecal fibroblast-like cells. Control follicles, in which an area opposite the germinal disc region was frozen 20 h before examination, had no degenerative features. These results provide further evidence that the germinal disc region is required for follicular growth.

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A Barua, H Michiue and Y Yoshimura

The aim of this study was to determine the changes in the population of major histocompatibility complex class II positive (MHC-II(+)) cells in ovarian follicles during the processes of follicular growth, postovulatory regression and follicular atresia in hens. Cryostat sections of ovarian stroma containing cortical follicles, small white follicles, the largest (F(1)) and third largest (F(3)) preovulatory follicles, postovulatory and atretic follicles of laying hens were prepared. The sections were immunostained for MHC-II molecules using mouse anti-chicken MHC-II monoclonal antibody and positive cells were counted using a computer-assisted image analyser under a light microscope. MHC-II(+) cells were localized in the theca layer of normally growing follicles including cortical follicles, small white follicles and F(3) and F(1) preovulatory follicles, whereas they were found in both the theca and granulosa layers in postovulatory and atretic follicles. The frequency of MHC-II(+) cells in the theca layer was significantly increased during follicular growth from cortical follicles to F(3) preovulatory follicles. Although the population of MHC-II(+) cells did not differ between F(3) and F(1) preovulatory follicles, it increased significantly in postovulatory follicles (P < 0.01). The population of MHC-II(+) cells was significantly greater in the theca layer of atretic follicles than in non-atretic follicles (P < 0.01). These results indicate that the antigen-presenting function via MHC-II increases in association with follicular growth. A marked increase in MHC-II(+) cells indicates that these cells may be involved in regression of postovulatory and atretic follicular tissues.

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W. M. Zheng, Y. Yoshimura and T. Tamura

The effects of age and gonadal steroids on the localization of immunocompetent cells, including antigen-presenting cells that contain the major histocompatibility complex (MHC) class II antigen, and T and B cells in the chicken oviduct were studied. Oviductal tissues were collected from laying and immature hens treated with diethylstilboestrol (an analogue of oestrogen) or progesterone. Cryostat sections of the tissues were immunostained for MHC class II, CD3 (T-cell antigen) and Bu-1 (immature B-cell antigen), and examined under a light microscope and an image analysis system. MHC class II+, CD3+ and Bu-1+ cells were observed in the mucosal epithelium and stromal connective tissue of both the laying and immature hens. MHC class II+ cells in the oviductal stroma appeared in association with oviductal development during sexual maturation and increased with ageing thereafter. The infiltration of CD3+ and Bu-1+ cells into the oviductal tissues increased in young laying hens compared with immature hens and decreased in old laying hens compared with young laying hens. Diethylstilboestrol increased the population of MHC class II+ and CD3+ cells in the stroma of the infundibulum and vagina, but had no significant effect on the population of Bu-1+ cells in the oviduct of immature hens. Progesterone increased the population of CD3+ cells in the stromal tissue of oviductal segments from all hens, and of Bu-1+ cells in the mucosal epithelium of the infundibulum and magnum, but had little effect on the frequency of MHC class II+ cells in the oviduct of immature hens. There were typically more immunocompetent cells in the infundibulum and vagina than in the other oviductal segments in laying hens and immature hens treated with sex steroids. These results suggest that local immunity in the chicken oviduct is enhanced during sexual maturation and possibly decreases during ageing. Gonadal steroids may play a significant role in the regulation of local immunity in the oviduct. The effects of oestrogen and progesterone on the influx of these immunocompetent cells into the oviduct differs among cell types and oviductal segments.

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Y. Yoshimura, T. Okamoto and T. Tamura

Proliferation of granulosa and thecal cells was analysed during ovarian follicular growth in laying Japanese quail. The birds were injected intraperitoneally with bromodeoxyuridine (BrdU) 10 or 4 h before ovulation, that is, before or after a preovulatory LH surge, respectively, and incorporation of BrdU by follicular tissues was detected immunocytochemically. Cells labelled with BrdU were seldom seen in the most immature follicles in the ovarian cortex, whereas many granulosa and thecal cells were labelled with BrdU in medium-sized white yolky follicles (approximately 13.3% and 14.4% in granulosa and theca layers, respectively). Ten and four hours before ovulation, the granulosa cells in the germinal disc and non-disc regions of the third largest yellow yolky follicle (F3) were labelled with BrdU (approximately 8.4% and 9.4% in germinal disc; 6.1% and 9.0% in the non-disc region), but only those in the germinal disc region were labelled (approximately 5.4% and 4.0%) in the largest yellow yolky follicle (F1). The percentage of thecal cells labelled with BrdU 4 h before ovulation was significantly higher than the percentage labelled 10 h before ovulation, and was higher in F3 (approximately 11.7%) than in Fl follicles (approximately 5.4%) 4 h before ovulation. These results show that proliferation of granulosa and thecal cells occurs in both germinal disc and non-disc regions in growing follicles, but when a follicle matures proliferation is reduced and in the case of granulosa cells it is restricted to the germinal disc region.

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A. Barua, Y. Yoshimura and T. Tamura

Immunoglobulins in the chicken ovary are important for transfer of immunity to chicks through the egg and for protection of the ovary from infection. The aim of this study was to examine the effects of ageing and oestrogen on the population of Ig-containing cells in the chicken ovary. The ovarian tissue of immature, young laying and old laying hens and that of immature birds treated with diethylstilboestrol (DES), progesterone or sesame oil (vehicle) was processed for paraffin wax sections. The sections were stained for IgG, IgM and IgAby an indirect immunostaining method and the population of cells positive for each Ig was analysed under a light microscope. The number of cells positive for IgG, IgM and IgA was significantly greater in the ovarian stromal tissue of young laying hens than in immature or old laying hens (P <0.01). The number of IgG- and IgM-positive cells in the thecal layer of primary follicles of young laying hens was significantly greater than that in immature and old laying hens (P < 0.01) and there were significantly more (P < 0.05) IgA-positive cells in young laying hens than in immature birds. The number of IgG-, IgM- and IgA-positive cells was significantly (P <0.01) greater in both the stromal tissue and the thecal layer of DES-treated birds than in the vehicle-treated birds. Progesterone had no significant effect (P < 0.05) on the population of Ig-positive cells. These results indicate that the number of Ig-positive cells increases as chickens mature and decreases with ageing, and that oestrogen may be involved in this process.

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A. Barua, Y. Yoshimura and T. Tamura

The role of macrophages in the function of the hen ovary has not yet been described, although these cells may be an important regulator of ovarian function in mammals. The aim of this study was to determine the changes in the frequency of macrophages during ageing and follicular atresia, and the effects of sex steroids on the macrophage population in the hen ovary. Cryostat sections of ovarian tissues of immature, young laying and old laying hens and those of immature hens treated with or without diethylstilboestrol (DES) or progesterone were immunostained for macrophage cells using mouse anti-chicken macrophage monoclonal antibody. Macrophages were observed under a light microscope and counted using a computer assisted image analyser. The frequency of macrophages in both the stroma and theca of primary follicles was significantly greater in young laying hens than in immature and old laying hens and these cells were more frequent in old laying hens than in immature hens (P < 0.01). Macrophages were more frequent in atretic follicles than in normal follicles (P < 0.01). The number of macrophages in both the stroma and theca of primary follicles of DES-treated birds was significantly greater than in those of progesterone-treated and control birds (P < 0.01). Progesterone had no significant effect on the population of macrophages. These results suggest that macrophages in the ovary increase in association with sexual maturation of birds and atresia of follicles and decrease during ageing. Oestrogen may be one of the factors that affect the population of macrophages in the hen ovary.

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Y. Yoshimura, T. Okamoto and T. Tamura

The aim of this study was to describe the temporal sequence of ultrastructural changes in the boundary between the preovulatory oocyte and its surrounding follicular wall during maturation induced by injection of LH. Female Japanese quail were injected with ovine LH (20 μg per bird) 10–12 h before the expected time of ovulation. The largest and second largest follicles were excised before or 1, 2, 4 or 6 h after injection. The oocyte and the surrounding follicular wall were processed for observations using light and electron microscopy. Before injection of LH, cytoplasmic projections of granulosa cells interdigitated with microvilli on the surface of the oocyte and formed spot desmosomes and gap junctions with the oolemma. Two hours after injection of LH, the germinal vesicles in the largest but not in the second largest follicles began to break down and membrane-bound vesicles increased in number and size in the surrounding germinal disc. The junctions between the oocyte surface and the granulosa cell projections started to dissociate and a perivitelline space began to develop, possibly as the result of an accumulation of fluids transported from the capillary sinus in the theca interna. The first maturation spindle was formed 4 h after injection of LH, whereas the first polar body and the second maturation spindle were formed 6 h after LH stimulation. These observations suggest that the dissociation of connections between the oocyte and granulosa cells 2 h after exposure to increased concentration of LH is the first process of oocyte maturation. The associated increase in number and enlargement of membrane-bound vesicles in the germinal disc may be involved in the activation of factors involved in oocyte maturation.

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Y. Yoshimura, T. Okamoto and T. Tamura

This study examined structural changes in oocyte and follicular wall during oocyte maturation in Japanese quails. The structures of the germinal disc and the surrounding follicular wall were observed by light and electron microscopy 25, 6 and 1 h before the expected time of ovulation. The germinal disc of the oocyte was located near the oocyte plasma membrane at 25 h before ovulation, and the germinal vesicle was located in the centre of the germinal disc. Numerous cytoplasmic elements, such as elongated membrane-bound vesicles, mitochondria and glycogen granules were also observed in the germinal disc. The surface of the oocyte made close contact with the cytoplasmic processes of the granulosa cells. Six hours before ovulation, fluid filled spaces formed between the oocyte and follicular wall. At 6 h before ovulation, the germinal disc was similar to that at 25 h before ovulation, whereas the oocyte and the granulosa cells were disconnected. Myelin bodies and dense bodies developed in the cytoplasmic processes of the granulosa cells, suggesting that lysosomal enzymes were activated. In the follicle at 1 h before ovulation, the second maturation spindle was located just beneath the surface of the oocyte, and the first polar body was in the perivitelline space. In the germinal disc, the membrane-bound vesicles were swollen and well developed. We suggest that, during the process of early oocyte maturation, the junctions between the oocyte and granulosa cells are disconnected, and factors that promote oocyte maturation may be activated in the germinal disc since the membrane-bound vesicles are developed.