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H Iida, T Kaneko, S Tanaka, and T Mori

The spermatozoa of the musk shrew, Suncus murinus, have a fan-like giant acrosome with a diameter of approximately 20 mm. The aim of this study was to investigate how this giant acrosome is constructed in the musk shrew spermatid and, in particular, how the Golgi apparatus involved in acrosome formation behaves. The behaviour of the Golgi apparatus was monitored by confocal laser scanning microscopy with antibody against a Golgi-associated Rab6 small GTPase. In the early Golgi phase, small Golgi units, the Golgi satellites, localized as a large aggregate in the juxtanuclear cytoplasm. As acrosome formation progressed, the Golgi satellites gradually dispersed, associated with proacrosomal vesicles and an acrosomal vesicle, and finally became distributed as multiple small units over the whole surface of an acrosomal cap in the round spermatid. The mode of acrosome formation in musk shrews was distinctly different from that in rats and mice, in which the Golgi apparatus remains as a single unit throughout acrosome formation. In musk shrews, the proacrosomal vesicles formed successively by the Golgi satellites coalesced, one after another, into a potential acrosomal vesicle. This process may result in further enlargement of the acrosome. The results of the present study indicate that Golgi satellites are necessary for the biogenesis and development of the giant acrosome in musk shrew spermatozoa.

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H Iida, T Kaneko, S Tanaka, and T Mori

The spermatozoa of the musk shrew, Suncus murinus, have a fan-like giant acrosome with a diameter of approximately 20 mm. The aim of this study was to investigate how this giant acrosome is constructed in the musk shrew spermatid and, in particular, how the Golgi apparatus involved in acrosome formation behaves. The behaviour of the Golgi apparatus was monitored by confocal laser scanning microscopy with antibody against a Golgi-associated Rab6 small GTPase. In the early Golgi phase, small Golgi units, the Golgi satellites, localized as a large aggregate in the juxtanuclear cytoplasm. As acrosome formation progressed, the Golgi satellites gradually dispersed, associated with proacrosomal vesicles and an acrosomal vesicle, and finally became distributed as multiple small units over the whole surface of an acrosomal cap in the round spermatid. The mode of acrosome formation in musk shrews was distinctly different from that in rats and mice, in which the Golgi apparatus remains as a single unit throughout acrosome formation. In musk shrews, the proacrosomal vesicles formed successively by the Golgi satellites coalesced, one after another, into a potential acrosomal vesicle. This process may result in further enlargement of the acrosome. The results of the present study indicate that Golgi satellites are necessary for the biogenesis and development of the giant acrosome in musk shrew spermatozoa.

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T. Takahashi, M. Kawashima, T. Yasuoka, and K. Tanaka

The molecular mass of the arginine vasotocin receptor in membrane fractions of the uterus (shell gland) of hens was estimated by autoradiography of [125I]-labelled arginine vasotocin binding protein after SDS-PAGE. A band of approximately 67 kDa was found in all of the samples taken from laying hens before and after oviposition and from nonlaying hens. In addition to this band, another band of approximately 95 kDa was found in samples from laying hens taken immediately before and after oviposition. Both bands were reduced by the presence of unlabelled arginine vasotocin or mesotocin, but the reduction was greater by arginine vasotocin than by mesotocin. These two bands were not reduced by chicken luteinizing hormone-releasing hormone-I (Gln8-GnRH), chicken luteinizing hormone-releasing hormone-II (His5,Trp7,Tyr8-GnRH)and chicken angiotensin-II (Val5-angiotensin-II). The appearance of the arginine vasotocin receptor of the larger molecular size may be related to oviposition in hens.

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M Nishihara, Y Takeuchi, T Tanaka, and Y Mori

The hypothalamic gonadotrophin-releasing hormone (GnRH) pulse generator governs intermittent discharges of GnRH into the pituitary portal circulation and, consequently, modulates the pulsatile pattern of gonadotrophin secretion. Electrophysiological correlates of pulsatile gonadotrophin secretion have been demonstrated in the mediobasal hypothalamus of monkeys, rats and goats by recording multiple unit activity. A temporal coincidence between characteristic increases in multiple unit activity and gonadotrophin pulses in the circulation is seen under a variety of physiological and experimental conditions in all three species examined, providing evidence that hypothalamic multiple unit activity originates in the GnRH pulse generator. During a preovulatory gonadotrophin surge induced by oestrogen in ovariectomized animals or occurring spontaneously in intact animals, GnRH pulse generator activity is decelerated, suggesting that it is not involved in generating the gonadotrophin surge. The gonadotrophin surge may be generated by an oestrogen-responsive neuronal complex intrinsically different from the GnRH pulse generator, the electrical operation of which remains unknown.

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K. Tanaka, T. Wada, O. Koga, Y. Nishio, and F. Hertelendy

The aim of this study was to produce viable chicks by in vitro fertilization and transfer of fertilized ova to the oviduct of recipient hens. Out of a total of 76 transferred ova, 53 were laid with fully calcified shells, 31 of which were fertile (58%). Despite the high rate of embryonic loss, six live chicks were hatched from 12 fertile ova exposed to 0.05 ml of semen (1:200 dilution). Nine healthy chicks were hatched from ten control ova which were recovered from the oviduct following artificial insemination and subsequent transfer to recipient hens. This experimental approach provides a useful model for production of transgenic chicks.

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M Tanaka, T Miyazaki, S Tanigaki, K Kasai, K Minegishi, K Miyakoshi, H Ishimoto, and Y Yoshimura

Prostaglandin F(2alpha) (PGF(2alpha)) is implicated in the process of luteal regression in many species. Treatment of rat luteal tissue with PGF(2alpha) increases the generation of reactive oxygen species. Since reactive oxygen species have been implicated in apoptosis, the present study was undertaken to determine whether reactive oxygen species play a role in the PGF(2alpha)-induced apoptosis of rat luteal cells. Rat luteal cells were loaded with 6-carboxy-2, 7'-dichlorodihydro-fluorescein (CDCFH) diacetate, di (acetomethyl ester), which can be oxidized by reactive oxygen species to yield CDCF, a fluorescent molecule, and the cells were treated with different doses of PGF(2alpha). Incubation with 100 micromol PGF(2alpha) l(-1) induced an increase in CDCF fluorescence (P < 0. 05). Treatment of cells with PGF(2alpha) for 48 h in serum-free medium induced a dose-dependent increase in cell death, and these cells exhibited the morphological characteristics typical of apoptosis, including condensed or fragmented nuclei and fragmentation of internucleosomal DNA. Pretreatment of these cells with ascorbic acid, N,N'-dimethylthiourea, or superoxide dismutase, which acts as an antioxidant or a radical scavenger, prevented the PGF(2alpha)-induced apoptosis. These results demonstrate that PGF(2alpha) produces reactive oxygen species and induces apoptosis in rat luteal cells, indicating that the reactive oxygen species may induce apoptotic cell death during luteolysis.

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K Iwahashi, N Kuji, T Fujiwara, H Tanaka, J Takahashi, N Inagaki, S Komatsu, A Yamamoto, Y Yoshimura, and K Akagawa

Syntaxin is an integral membrane protein that is involved in membrane fusion. The exocytosis of the contents of cortical granules, secretory vesicles located in the cortex of an egg, modify the extracellular environment to block additional spermatozoa from penetrating the newly fertilized egg. The aim of this study was to characterize syntaxin expression in mouse oocytes, and to determine the specific isoform that is expressed. Syntaxin was demonstrated in the mouse ovary and in mouse oocytes by both western blot and reverse transcription-polymerase chain reaction analyses. Syntaxin 4 was specifically expressed in metaphase II oocytes. Syntaxin was also immunolocalized within metaphase II oocytes and one-cell embryos with pronuclei using laser scanning confocal microscopy. In metaphase II oocytes, syntaxin was located on the plasma membrane and in the cortex, where cortical granules are present, but was not seen at sites free of cortical granules. In one-cell embryos, no cytoplasmic region was free of syntaxin immunoreactivity. Immunoelectron microscopy detected syntaxin on both the plasma membrane and the vesicle membranes in mouse metaphase II oocytes. In conclusion the results indicate that syntaxin 4 co-localizes with cortical granules and participates in membrane fusion and exocytosis during the cortical reaction.