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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

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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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.

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

The effects of sexual maturation and gonadal steroids on the localization of immunoglobulin-positive cells in chicken oviducts were studied. Oviductal tissues were collected from laying hens and chicks treated with stilboestrol (DES, an analogue of oestrogen) or progesterone. Paraffin wax sections of the tissues were immunostained for IgG, IgM and IgA, and the frequency of cells staining positive was examined using an image analysis system. Some of the cells in the mucosal epithelium and plasma cell-like cells in the stroma of the oviduct stained positive for IgG, IgM or IgA. In the mucosal epithelium of laying hens, there was a significantly greater number of IgG-positive (IgG+) cells in the shell gland than in the infundibulum, magnum and isthmus, more IgM+ cells in the magnum than in the infundibulum, and more IgA+ cells in the magnum than in the other segments of the oviduct with the exception of the vagina. The frequency of IgG+ and IgM+ cells in the mucosal epithelium of all oviductal segments and IgA+ cells in the magnum, isthmus and vagina was significantly higher in laying hens than in immature birds. In the subepithelial stroma of laying hens, there was a significantly greater population of IgG+ cells in the infundibulum and vagina than in the magnum and isthmus, more IgM+ cells in the infundibulum than in the magnum, and more IgA+ cells in the uterovaginal junction and vagina than in the magnum and isthmus. The frequency of IgG+, IgM+ and IgA+ cells in the subepithelium of infundibulum, uterovaginal junction and vagina was significantly greater in laying hens than in immature birds. The number of IgM+ cells in all oviductal segments and of IgA+ cells in the magnum of the mucosal epithelium of the chicks treated with DES increased significantly compared with those of control chicks. In addition, the number of IgG+ cells in the shell gland and vagina and of IgM+ cells in the vagina of the stroma of DES-treated birds were increased. Treatment of immature birds with progesterone had no effect on the localization of Ig+ cells in the oviduct except for a decrease in the number of IgM+ cells in the shell gland. These results suggest that the local immunity in the oviduct develops during sexual maturation, possibly under the control of oestrogen.

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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.