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Background and Aims: Bromoacetic acids are a by‐product of water ozonation and dibromoacetic acid (DBAA) in particular, which is a by‐product of disinfection, inhibits male reproductive functions. In order to understand its effects, the spermatozoa and testes of mice were exposed to DBAA.

In mammals, circadian rhythms are driven by a pacemaker located in the suprachiasmatic nuclei (SCN) of the hypothalamus. The pacemaker is composed of an ensemble of multiple, single-cell oscillators in the SCN. We measured arginine-vasopressin (AVP) release in organotypic SCN slices. The SCN slice culture showed circadian oscillation of AVP release with a period length (+/- SEM) of 23.84 +/- 00.03 h. This period is very similar to the one we previously reported in dispersed SCN cultures and is also close to that of behavioural rhythms. When the ventral part was removed by a surgical cut across the slice in the horizontal plane, however, the period became shorter (23.22 +/- 00.08 h). On the other hand, the removal of the dorsal part did not affect period length. These results suggest that the oscillators in ventral and dorsal cells contribute differently to period length and that the dorsal oscillators are entrained by the ventral ones to form a single integrated oscillator.

Bromoacetic adds are a by-product of water ozonation and dibromoacetic acid (DBAA) in particular, which is a by-product of disinfection, inhibits male reproductive functions. In order to understand its effects, the spermatozoa and testes of mice were exposed to DBAA. Twelve-week-old ICR mice were exposed to 10 p.p.m. DBAA. They were examined in regards to effects on the weights of body, testis and epididymis, the histological changes of tesits and the protein expression in testis. Neither the bodyweight nor the weights of the testis and epididymis of the exposed mice was affected, but approximately 13% of spermatozoa obtained from the cauda epididymis were motile with a drop-shaped head, and structures resembling residual bodies were found in the testis. Moreover, the expression of two testis proteins was changed by exposure to DBAA. It was likely that DBAA inhibited male reproductive functions by disturbance of spermatogenesis via change of protein expression.

In our previous studies (Fujinoki et al., 2001, 2003), we reported that two types of 36 kDa proteins, designated 36K-A protein and 36K-B protein, obtained from hamster sperm flagella, are associated with motility activation and phosphorylated in a cAMP-dependent manner at serine residues. In the present experiments, we focused on the hamster (Mesocricetus auratus) 36K-A protein, which was analyzed by peptide mass finger printing and amino acid sequencing. The results suggest that 36K-A protein is a pyruvate dehydrogenase E1 component beta subunit lacking the N-terminal 30 amino acids. Moreover, our results suggest that 36 K-A protein is localized in the fibrous sheath of the principal piece of hamster spermatazoa.

In our previous paper [M. Fujinoki et al. (2001) BIOMED: Res. 22, 45-58], we reported that two types of 36-kDa protein, which were designated as 36K-A protein and 36K-B protein, obtained from hamster sperm flagella were phosphorylated at serine residues associated with the regulation of motility activation. In the present experiments, it was suggested that these two types of 36-kDa protein were phosphorylated in a cAMP-dependent manner associated with motility activation of hamster spermatozoa. Because the 36K-B protein was the most intensely phosphorylated in a cAMP-dependent manner, attempts were made to further characterize it. The 36K-B protein was assumed to be localized in the middle piece. The localization of the 36K-B protein was the same as that of the 36-kDa protein reported in our previous paper [Y. Si et al. (1999) Mol. Reprod. Dev. 52, 328-334]. In order to identify the 36K-B protein, it was analyzed by peptide mass finger printing and amino acid sequencing. The results suggested that the 36K-B protein was a pyruvate dehydrogenase E1 component beta subunit and a component of the mitochondrial sheath of the middle piece.

To evaluate the biological response of the horse to endotoxemia, a sublethal amount of lipopolysaccharide (LPS) (10 μg/kg) was given to two Thoroughbreds in two doses 24 hr apart. Each infusion initially produced a significant increase or decrease in rectal temperature, an increase in respiratory rate and heart rate, and a marked decrease in white blood cell count (WBC). The horses subsequently showed signs of shock, characterized by extreme coldness of the skin of lower limbs, as well as cyanosis of the visible mucosae, oliguria accompanied by proteinuria, and abnormalities in gastrointestinal function. The clinical signs of equine endotoxemia may be manifested by the clinical condition of the systemic inflammatory response syndrome (SIRS): two or more of the above signs, such as high or low rectal temperature, increased heart rate and respiratory rate, and leukopenia. Pathobiological responses such as a severe decrease in the number of platelets, hypoglycemia, metabolic acidosis, disorders of hemostasis accompanied by cyanosis of the visible mucosal membranes, proteinuria and oliguria, and abnormal gastrointestinal function, may be manifestations of pathobiological conditions ranging from disseminated intravascular coagulation (DIC) to multiple organ dysfunction syndrome (MODS). Therefore, the clinicopathological responses in equine endotoxemia suggest the nature of SIRS elicited by hypercytokinemia progressing into septic shock.

Thyroid hormone (TH) plays an important role in brain development. The rodent cerebellum has been an excellent model for the study of the molecular mechanisms of TH action in brain. However, most studies have utilized the rat rather than the mouse. Considering the usefulness of mice with regard to diverse genetic models, the study of TH effect on mouse cerebellar development is needed. Thus, we examined the effect of perinatal hypothyroidism on the expression of neurotrophin-3 (NT-3) and brain-derived neurotrophic factor (BDNF) genes, which play critical roles in cerebellar development. Newborn mice were rendered hypothyroid by administering methimazole and perchlorate in drinking water to their mothers. The growth of hypothyroid mice was retarded, which was reversed by daily thyroxine administration. NT-3 and BDNF gene expression was depressed in the perinatal hypothyroid cerebellum. Furthermore, the expression of retinoid-receptor-related orphan nuclear hormone receptor-alpha (RORalpha), an orphan nuclear receptor that plays critical roles in Purkinje cell development, was also decreased. Morphologically, disappearance of the external granule cell layer was retarded and arborization of Purkinje cell dendrite was decreased, events that were also observed in hypothyroid rats. These results indicate that the mouse cerebellum is comparable to the rat cerebellum as a model for the examination of the molecular mechanisms of TH action in brain development.

We tested the hypothesis that frequency of intense exercise during training would affect the likelihood of bucked shins complex developing in young horses. The relative prevalence of bucked shins were examined in two groups of Anglo-Arabian horses that trainined by different training programs. One group (6 horses) exercised once per week at maximal speed, the other group (7 horses) exercised twice per week at maximal speed. They ran on similar track surface conditions on the same track. Although the group exercised twice per week at maximal speed had a prevalence, there was no significant difference in the incidence of bucked shins between the two groups. It suggest that the number of maximum gallop than the duration of intense exercise. Pathomorphogenetically, there was suggestion that focal radiolucent areas, indicating focal remodeling regions consisting of a focal conglomerate of irregular-sized osteons, at the site of the middle layer in the cortical bone beneath periosteal bony proliferation might play a role as a precursor lesion for the occurrence of otitis of the third metacarpal bone (Mclll).

Proliferation of hepatocytes in liver regeneration is regulated by humoral and neuronal factors. Humoral factors include hormones and growth factors (mitogens). The neuronal factors, however, have not yet been identified. Factors that amplify the effect of complete mitogens (epidermal growth factor, transforming growth factor a and hepatocyte growth factor) are termed co-mitogens, which include vasopressin, angiotensin II, noradrenaline, neurotensin and neuropeptide-y. We have surveyed various other neuropeptides and gastrointestinal hormones that may have a co-mitogenic effect on rat hepatocytes using a complete serum-free primary culture system. Pancreatic polypeptide at 10 - 9 to 10 - 6 M amplified DNA synthesis stimulated by a low concentration of EGF (1 ng/mL) by 4- to 5-fold. Neuropeptide Y or peptide YY that have a structural similarity to pancreatic polypeptide did not amplify DNA synthesis. A similar amplification was found for secretin (10 - 1 0 to 10 - 7 M). These results indicate that pancreatic polypeptide and secretin can be regarded as novel co-mitogens, and suggest that these peptides and other co-mitogenic neuropeptides, most of them are found also in neurons, are candidate neuronal factors in hepatocyte proliferation in vivo, i.e. liver regeneration.

Mammalian circadian pacemaker is located in suprachiasmatic nuclei (SCN) of the hypothalamus. The pacemaker is entrained by light-dark cycle; the photic information is transmitted primarily via the retino-hypothalamic tract (RHT). The main neurotransmitter of the tract is glutamate. RHT fibers end on the ventrolateral part of the nucleus, where vasoactive intestinal peptide (VIP)-immunopositive neurons are localized. They send their axons into dorsomedial SCN, where most of the vasopressinergic (AVP) neurones are located. The AVP neurons retain the clock-like properties in vitro. Vasopressin release from the cultured neurons shows circadian rhythm peaking in the middle of subjective day. VIP induces phase-shifts of the rhythm, magnitude and direction of the shift depending on timing of the application. VIP applied 6-12 h before the peak of vasopressin rhythm induces advances, application 4-8 h after the peak induces delays. The lowest concentration required to induce the phase-shift is 30 nM, further increase of the concentration does not affect the magnitude of the shift. In contrast, glutamate has no effect on the phase of vasopressin rhythm, although in high concentrations it transiently stimulates vasopressin release. The data indicate that the vasopressinergic cells in the SCN contain circadian oscillators, whose rhythms run mutually synchronized in our cultures. VIP acts directly on the vasopressinergic cells to shift the phase of their pacemakers; glutamate has no such effect presumably because in vivo it acts through the VIP-ergic cells but the neuronal network is altered after the dissociation of the cells.

Oxidant stress has been implicated in the pathogenesis of Parkinson's disease. To test the oxidant stress hypothesis of dopaminergic degeneration, age-related changes in the mesostriatal dopamine neuron system were compared between zitter mutant rats which have abnormal metabolism of oxygen species in the brain and Sprague-Dawley rat as a control using the neurochemistry and immunohistochemistry. Dopamine content in the caudate-putamen, nucleus accumbens and olfactory tubercle of zitter rats decreased significantly with age, and was lower than that found in corresponding age-matched controls. In the zitter rats, the reduction of dopamine was more prominent in the caudate-putamen than in the nucleus accumbens and olfactory tubercle. A characteristic decline of tyrosine hydroxylase-immunoreactive fibers in the caudate-putamen of the zitter rat was also observed. In the dorsolateral caudate-putamen, reduction of tyrosine hydroxylase-immunoreactive fibers was observed in the matrix-like area, whereas in the ventromedial caudate-putamen the reduction occurred in the patch-like areas. Degeneration of tyrosine hydroxylase-immunoreactive fibers which was characterized by swollen varicosities and clustered fibers was observed in the caudate-putamen and nucleus accumbens and preceded loss of normal tyrosine hydroxylase-immunoreactive fibers in the caudate-putamen. Thus, the depletion of dopamine in the terminal areas is related to axonal degeneration. However, there was no degenerative tyrosine hydroxylase-immunoreactive fibers in the olfactory tubercle at any examined age, but reductions of tyrosine hydroxylase-immunoreactive fibers and dopamine contents were noted in the olfactory tubercle after four months-of-age. Since the zitter rats have an abnormal oxygen metabolism, the degeneration of tyrosine hydroxylase-immunoreactive fibers could result from an accumulation of superoxide species. The present results provide support for the oxidant stress hypothesis of dopaminergic neuronal degeneration and further indicate the region-specific vulnerability of the nigrostriatal dopamine system.

A distribution of abnormal catecholaminergic and serotonergic fibers was in the spinal cord of 12-month-old zitter mutant rats which are characterized by abnormal metabolism of superoxides studied using antisera for tyrosine hydroxylase (TH) and serotonin. Similar to our previous report [13], abnormal serotonergic fibers characterized by swollen varicosites were observed mainly in the anterior and lateral columns of the cervical and lumber segments of zitter rats. On the other hand, abnormal TH-immunoreactive fibers with swollen varicosities were located in the anterior and lateral funiculus of lumbar and sacral segments of these mutant rats. The present results suggest that the degeneration patterns differ between catecholaminergic and serotonergic fibers in aged-zitter rats.

The serotonin neuron system was studied using immunohistochemical and neurochemical techniques in zitter mutant rats aged 1-14 months, which are characterized by abnormal metabolism of superoxides. The morphology of the serotoninergic neuron system and the serotonin level in the zitter rat were compared to those of age-matched Sprague-Dawley (SD) rats. Up to age 4 month, the density and distribution of serotoninergic fibers in the zitter rat brain were similar to those of control rats. However, several serotoninergic fibers with abnormal morphology, characterized by swollen varicosities, were observed in the cerebral cortex and caudate putamen of 6-month-old zitter rats. The density and distribution of these fibers in other regions of the brain were similar to those of control rats. The abnormal serotoninergic fibers increased in number and extended into other regions of the brain such as the thalamus, hippocampus, and vestibular nucleus. On the other hand, the density of normal serotoninergic fibers decreased throughout the brain of the 14-month-old zitter rat. Abnormal serotoninergic fibers have also been reported in the brain of normal older (24 months) SD rats. Neurochemical analysis revealed lower levels of serotonin, and 5-hydroxyindoleacetic acid, in all cortical areas (prefrontal, parietal, and occipital cortices), the caudate putamen, and the hippocampus of 12-month-old zitter rats. Levels differed significantly in the parietal cortex and hippocampus between the zitter and SD rats. Based on the morphological and neurochemical similarities, the present results suggest that age-related degeneration of serotoninergic fibers occurs in the zitter rat brain. Furthermore, degeneration of serotoninergic fibers appears to be induced by superoxide species. Thus, the zitter rat may provide a good model for studying the neurotoxic effects of superoxide species on the serotoninergic neuron system.

A dissociated cell culture system has been developed for the suprachiasmatic nucleus (SCN), in which release of vasopressin showed a clear circadian oscillation. The oscillation peaked at early subjective day and appeared within one day in culture. This system may provide a valuable model for the study of cellular and molecular mechanisms of the mammalian circadian pacemaker.

An efficient modified adeno system to express foreign genes to the central nervous system was developed recently. This modified recombinant adenoviral vectors can be used successfully to deliver lacZ to the hypothalamic suprachiasmatic nucleus (SCN) which is composed of mammalian clock pacemaker. The expression of lacZ in the primary culture of SCN was dose-dependent and higher enough in nearly 100% of these cells. We also showed that viral toxicity and lacZ overexpression had no serious effects on the rhythmic expression of arginine vasopressin (AVP) release from the SCN cell culture.

Tanaka S, Koibuchi N, Ohtake H, Ohkawa H, Kawatsu T, Tadokoro N, Kumasaka T, Inaba N, Yamaoka S. Regional comparison of prolactin gene expression in the human decidualized endometrium in early and term pregnancy. Eur J Endocrinol 1996;135:177–83. ISSN 0804–4643 Prolactin (PRL) is known to be expressed in the decidualized human endometrium and secreted into amniotic fluid. Although the site of synthesis of endometrial PRL is known to be the decidual cells, the difference in PRL gene expression within each area of decidua, i.e. decidua basalis, decidua parietalis and decidua capsularis, during pregnancy is not clear. We have applied an in situ hybridization histochemistry technique using a radiolabeled RNA probe to compare the difference in expression of PRL gene within each area of the decidualized endometrium. Specific hybridization signals were distributed over the decidual cells in early and term pregnancy. More intense hybridization signals were always detected in the tissues of early pregnancy than in those of term pregnancy. In the decidua capsularis of early pregnancy, labeled cells were concentrated close to the amniotic cavity, whereas cells were concentrated close to the maternal surface of the fetal membrane in term pregnancy. In the decidua parietalis, almost all decidual cells were labeled, but no specific labeling was seen in the endometrial glands or capillary endothelium in both groups. In the decidua basalis, most decidual cells showed hybridization signals whereas no hybridization signal was seen over the trophoblast cells. These results show that there are regional and periodic differences in PRL gene expression in the decidual cells during pregnancy. Noriyuki Koibuchi, Department of Physiology, Dokkyo University School of Medicine, Mibu, Tochigi 321-02, Japan

The morphological features and distribution of serotonergic fibers in the spinal cord of 7-and 12-month-old zitter mutant rats which are characterized by abnormal metabolism of superoxides were examined immunohistochemically. In the zitter rat spinal cord, several aberrant serotonergic fibers characterized by swollen varicosities were intermingled with normal serotonergic fibers. These aberrant fibers increased in number by aging. Serotonergic neuron system seem to be vulnerable to superoxide radicals. Thus, these aberrant serotonergic fibers may reflect the degeneration by superoxide radicals. These aberrant fibers were never found in young adult or 12-month-old SD rats. The appearance of these fibers has been reported in the aged (30 months) rat spinal cord. Based on the morphological similarity, the present study indicates the early onset of age-related change in serotonergic fibers in the spinal cord of zitter rats.

Prolactin (PRL) is known as an anterior pituitary hormone. On the other hand, PRL is also produced in the human decidualized endometrium. The physiological role and site of action of endometrial PRL have not yet been clarified. This study was designed to investigate the localization of PRL receptor (PRL-R) gene-expressing cells in the human decidualized endometrium using in situ hybridization histochemistry. Sense and antisense 35S-labeled RNA probes for human PRL-R mRNA were hybridized with cryostat sections of human decidua, which were obtained from patients undergoing therapeutic abortion at 8-10 weeks of gestation. Hybridization signals for PRL-R mRNA were seen over the decidual cells. No labeled cells were seen in the chorion, amnion, or trophoblast. Comparing the localization of PRL-R gene-expressing cells to that of PRL gene-expressing cells using adjacent sections, their distributions were quite similar. These results indicate that not only PRL but also PRL-R transcripts are located in the decidual cells.

Early development of the central nervous system is influenced by several hormones including thyroid hormone. This study was designed to clone the gene whose expression is changed in association with perinatal hypothyroidism in the rat cerebellum. Rats were sacrificed at 15 day-old postnatal age (P15) and their cerebella were removed. Poly (A)+ RNA was extracted to construct a cDNA library using lambda gt 10 cloning vector. Differential plaque screening was then performed using 32P-labeled antisense cDNA synthesized from poly (A)+ RNA of the methimazole-treated (hypothyroid) P15 rat cerebellum (hypothyroid probe), and of the euthyroid P15 rat cerebellum (euthyroid probe). The clones, which hybridized strongly to the euthyroid probe and weakly or not at all to the hypothyroid probe, were isolated. Sequence analysis of these clones revealed that all isolated clones encode cytochrome c oxidase subunit I (COX I), which is located in the mitochondrial DNA. The decrease in COX I gene expression was not seen in the animals, which received methimazole treatment and daily replacement of thyroid hormone. In situ hybridization detection showed not only overall decrease in COX I gene expression but also change in distribution of hybridization signal in the cerebellar cortex of hypothyroid rat. Such change was not observed in the T4-replaced animals. Based on the evidence that thyroid hormone greatly influences brain development, the results of the present study indicate that the terminal enzyme of mitochondrial respiratory chain, COX I is one of the important target molecules regulated by thyroid hormone in the newborn rat cerebellum.

We have developed a cell culture system for the rat suprachiasmatic nucleus, in which a clear circadian oscillation of vasopressin release was observed. Using this culture system, the effect of anisomycin, an inhibitor of protein synthesis, on the circadian rhythm was studied. A phase-delay of more than 15 h could be produced by a 6-h anisomycin pulse. The magnitude of the phase-shift was dependent on the circadian time of the drug treatment and on its dose. The phase-response curve was similar to the response curves that have been measured for protein synthesis inhibitors in other organisms. These results strongly suggest that protein synthesis may be involved in the generation of circadian rhythms in mammals.

In order to detect the localization of c-erbA alpha 2 mRNA in the hypothalamus and its surrounding structures, in situ hybridization histochemistry was performed in the adult male rat. Sections through the hypothalamus were hybridized with a [3H]-labeled RNA probe complementary to c-erbA alpha 2 mRNA and then coated with photographic emulsion. Autoradiograms were then developed and localization of the hybridization signal was detected under a light microscope. Hybridization signal was detected throughout the hypothalamic and extrahypothalamic structures such as the paraventricular hypothalamic nucleus (PVN), ventromedial nucleus (VMN), arcuate nucleus (Arc), amygdala (Am), habenula (Hb), paraventricular thalamic nucleus (PVT) and hippocampus (Hipp). No hybridization signal was detected in the section which was hybridized with the sense probe. These results not only confirm previous findings in the PVN, Hb, PVT and Hipp, but also extend the finding to the VMN, Arc and Am, in which cells expressing c-erbA alpha 2 primary transcript were concentrated. These new findings may provide an important aid to the understanding of the roles of thyroid hormone in the function of the central nervous system.

In the seaward migrating chum salmon, immunocytochemical and in situ hybridization techniques revealed isolated GnRH neurons at the base of the nasal epithelium, along the nervus terminalis and as ganglia at the rostroventral (gROB) and caudalmost (GT) olfactory bulb. A novel GnRH ganglion was seen at the cribriform bone (gCB). GnRH immunoreactivity but not the hybridization signal was detected in the midbrain neurons. During the migratory period, there were trends towards an increase in GnRH mRNA in the gCB and the gROB and a significant surge in plasma thyroid hormones was also evident. Therefore, we hypothesise thyroid hormones might be crucial for the increased tendency of GnRH expression and the migratory behavior of chum salmon.

The expression of the ornithine decarboxylase (ODC) gene in the mouse kidney following testosterone treatment was examined using in situ hybridization histochemistry. Testosterone (n = 5) or vehicle (n = 5) was subcutaneously injected (1 mg/animal) into male BALB/c mice (8 weeks in age) 14 h before sacrifice. Animals were sacrificed under ether anesthesia, their kidneys were removed and immediately frozen in liquid nitrogen. Frozen sections (10-microns-thick) were cut on a cryostat. Sections were hybridized with 35S-labeled sense or antisense RNA probe. The hybridization continued for 24 h at 50 degrees C and emulsion autoradiography was subsequently performed. A marked increase in ODC mRNA was exclusively detected in the proximal tubule of the renal cortex in the testosterone-treated animals. The hybridization signal was greater in the outer portion of the proximal tubule than in the inner portion. No significant hybridization signal was detected either in the distal tubule, renal corpuscle or peritubular tissues. These results indicate that testosterone induces the expression of the ODC gene in the proximal tubule of the renal cortex, leading to the increase in ODC activity in the same region.

We have developed a suprachiasmatic nucleus (SCN) cell culture system to study the cellular and molecular bases of the mammalian circadian pacemaker. The SCN regions were dissected from 4- to 6-day-old rat pups and dissociated cells were cultured in a defined medium. In all the cultures, the release of vasopressin showed clear circadian oscillation, which appeared within a few days in culture and lasted for more than a month. The peak of vasopressin release was observed at subjective day. These findings suggest that this culture system provides a valuable model for elucidating the mechanism of the circadian pacemakers.

This work was designed to examine the effect of butylphthalide (BuPh) on the duration of anesthesia induced by intraperitoneal injection of pentobarbital in adult male ddy mice. Anesthetic period was measured by the duration of loss of righting reflex. Administration of sodium pentobarbital (50 mg/kg, i.p.) induced anesthesia lasting for about 28 min. Inhalation of BuPh during anesthesia markedly increased the duration of anesthesia. When the nasal cavity of animal was infused with 5% zinc sulfate in 0.5% NaCl 2 days prior to BuPh inhalation, such an increase was not observed, whereas infusion with 0.5% NaCl alone did not abolish the effect of BuPh

Plant-derived odorants promote good feeling, refresh spirits and sometimes relieve various stresses in humans. Physiological and psychological effects of plant-derived volatile chemicals have long been acknowledged in folk medicine and aromatherapy. Recent evidence from animal experiments suggests that these plant-derived chemicals affect various animal behaviors via modulating neural or humoral mechanisms (Torii et al., 1988; Sano et al., 1998; Ilmberger et al., 2001; Akutsu et al., 2002; Nakashima et al., 2004). However, the majority of those animal experiments were performed under unusual environmental conditions (various stresses, anesthesia). Therefore, we examined the effects of these odorants on sleep– wakefulness and circadian rhythm in freely moving animals. We used two volatile substances: α-pinene and mixture of green odor (nhexenal and n-hexenol).

In mammals, circadian rhythms are driven by a pacemaker located in the suprachiasmatic nuclei (SCN) of the hypothalamus. The pacemaker is composed of an ensemble of multiple, single-cell oscillators in the SCN. We measured arginine-vasopressin (AVP) release in organotypic SCN slices. The SCN slice culture showed circadian oscillation of AVP release with a period length (+/- SEM) of 23.84 +/- 00.03 h. This period is very similar to the one we previously reported in dispersed SCN cultures and is also close to that of behavioural rhythms. When the ventral part was removed by a surgical cut across the slice in the horizontal plane, however, the period became shorter (23.22 +/- 00.08 h). On the other hand, the removal of the dorsal part did not affect period length. These results suggest that the oscillators in ventral and dorsal cells contribute differently to period length and that the dorsal oscillators are entrained by the ventral ones to form a single integrated oscillator.

In our previous paper [M. Fujinoki et al. (2001) BIOMED: Res. 22, 45-58], we reported that the 58-kDa protein obtained from hamster sperm flagella was phosphorylated at serine residues in association with the start of motility. In the present experiments, we identified and localized the 58-kDa protein. The 58-kDa protein was assumed to exist in the acrosomal region domain of the sperm head and the whole sperm flagellum. In particular, a large amount of 58-kDa protein was localized in the equatorial segment of the acrosomal region domain of the sperm head and the middle piece of the sperm flagellum. In the next step, the 58-kDa protein was identified by peptide mass finger printing and LC-MS/MS analysis. The results suggested that the 58-kDa protein was ATP synthase H(+) transporting F1 beta, which is one of the mitochondrial components. Therefore, it is likely that the 58-kDa protein is associated with ATP production in the mitochondrial sheath in the middle piece of the sperm flagellum, and H(+) transport in the sperm head and the sperm flagellum except for the middle piece, since ATP synthase also acts as an H(+) pump.

We have studied melatonin effects on vasopressin release from dispersed cells of the rat suprachiasmatic nuclei (SCN). The release follows a circadian rhythm peaking during the day and decreasing at night. Melatonin inhibits the spontaneous increase and accelerates the decrease of vasopressin release. Melatonin also inhibits vasopressin release induced by vasoactive intestinal peptide (EC50=0.4 nM). The inhibition of vasopressin release correlates with the known inhibitory effect of melatonin on spontaneous neuronal activity in SCN.

The thyroid hormone plays a critical role in normal development of the mammalian central nervous system. This study was designed to examine the effect of perinatal hypothyroidism on ontogenic change in cytochrome c oxidase subunit I (COX I) gene expression in the rat cerebellum by using quantitative in situ hybridization histochemistry (ISH). Newborn rats were rendered hypothyroid by continuous administration of methimazole in the mothers' drinking water. The pups were then killed by decapitation on 1, 5, 10, 15, 20, and 30 days after birth (P1, P5, P10, P15, P20, and P30). Their cerebella were removed, and frozen sections were cut and processed for ISH with 35S-labeled RNA probe for COX I messenger RNA. After hybridization, emulsion autoradiography was performed. The numbers of grains within the external granule cell layer, molecular layer, and internal granule cell layer were then counted. A significant decrease in grain density was detected in the hypothyroid animal in all these areas on P5, P10, and P15. On P15, in the molecular layer, a greater hybridization signal was detected in the inner portion than in the outer portion in the euthyroid animal. No such difference was seen in the hypothyroid animal. Daily T4 treatment for 15 days restored the effect of methimazole treatment. The significant effect of perinatal hypothyroidism on COX I gene expression was not detected after P20. These results indicate that altered thyroid states affect the COX I gene expression in the cerebellar cortex during development, suggesting that the COX I gene is one of the key genes regulated by the thyroid hormone and plays an important role in the morphogenetic changes observed in the perinatal hypothyroid cerebellum.

D-Amino acid oxidase (DAO), which catalyzes oxidative deamination of D-amino acids, is known to be highly expressed in the kidney. This study was designed to examine the localization of DAO mRNA in the mouse kidney using in situ hybridization histochemistry (ISH). For comparison, ISH for mRNA of ornithine decarboxylase (ODC), which is also highly expressed in the mouse kidney, was simultaneously performed. Adult, male mice which received 1 mg of testosterone propionate or vehicle injection, were sacrificed 14 h after injection and their kidneys were removed and processed for ISH. Hybridization signals for both mRNAs were exclusively located over the epithelial cells of the proximal tubule in the vehicle-treated animals. Signals for the DAO mRNA were observed at nearly the same hybridization intensity throughout the proximal tubule, whereas hybridization signals for the ODC mRNA were observed exclusively in the pars convoluta. Following testosterone treatment, ODC mRNA in the pars convoluta was expressed with a stronger intensity than that in the vehicle-injected animals. ODC mRNA was also expressed in the pars recta with a weaker intensity than in the pars convoluta. On the other hand, DAO mRNA expression was little affected by testosterone treatment. These results indicate that, although both genes are possibly expressed in the same cells, the expression of these genes is regulated by different mechanisms.

The prolactin (PRL) gene is known to be expressed not only in the anterior pituitary but also in the decidualized human endometrium. This study was designed to detect the site of synthesis of PRL during pregnancy by in situ hybridization histochemistry. Decidual and trophoblast tissues from early pregnancy were obtained from patients undergoing therapeutic abortion at 8-10 weeks of gestation. Term placentae were obtained from patients with uncomplicated deliveries at 38-40 weeks. Sections of these tissues were hybridized with 35S-labeled RNA probe complementary to human PRL mRNA. Specific hybridization signals were distributed over the decidual cells in early and term pregnancy. In the decidua capsularis of early pregnancy, labeled cells were concentrated close to the amniotic cavity, although decidual cells were distributed evenly. In the decidua parietalis, almost all decidual cells were labeled, but no specific labeling was seen in the endometrial glands or capillary endothelium. In the decidua basalis, greater signals were always detected over the decidual cells in early pregnancy than in term pregnancy, when sections, which were hybridized with the same probe and exposed simultaneously, were compared. No specific hybridization was detected in the trophoblast cells. These results not only confirm that PRL is specifically synthesized in the decidual cells but also indicate that there are regional and periodical differences in PRL gene expression in the decidual cells during pregnancy.

To examine thyroid hormone regulation of c- erb Aα2 mRNA expression in the parvo-cellular region of the paraventricular nucleus of the rat hypothalamus (pPVN), quantitative in situ hybridization was performed using 3 H-labeled probe complementary to c- erb Aα2 mRNA. Thyroidectomy induced a significant increase in the number of silver grains overlying the cytoplasm in the pPVN relative to sham-operated controls. This effect was prevented by daily injection of thyroxine. These results indicate that hypothyroidism induced an increase in cellular c- erb Aα2 mRNA level in the pPVN.

The circadian change in the number of the Fos-like immunoreactive (IR) cells in the suprachiasmatic nucleus (SCN) was examined in the adult male rats, whose eyeballs were enucleated two months before sacrifice. Their circadian rhythms were determined by their locomotor activity. They were sacrificed in the middle of the active phase or inactive phase. Then brain sections were cut for immunocytochemistry for Fos. A marked increase in the number of the Fos-like IR cells was observed in the inactive phase in the SCN, whereas no such increase was observed in the supraoptic nucleus. These results indicate that, in the SCN, Fos expression was changed with endogenous circadian rhythm in the free-running rat.

The effect of testosterone on the activity of ornithine decarboxylase (ODC), its protein level and immunocytochemical distribution were examined in the mouse kidney. Male BALB C mice at 8 weeks of age were used throughout. Fourteen hours before death, they received a subcutaneous injection of testosterone (1 mg/animal) or solvent to measure renal ODC activity or to detect the distribution of ODC immunoreactivity in the kidney. Renal ODC activity and the content of the enzyme were markedly increased after testosterone treatment. Histologically, few cells that were obviously immunoreactive to ODC were observed in the control animals and in the testosterone-treated animals a marked increase in ODC immunoreactivity was observed only in the cortex. ODC immunoreactive cells were located diffusely in the proximal tubule. In the pars recta, cells were stained weakly and homogeneously, while in the pars convoluta, the luminal surface of the cells showed stronger immunoreactivity. Moreover, many granule-like particles that were strongly ODC immunoreactive were observed inside the lumen of the pars convoluta. These results show that testosterone treatment induces an increase in ODC content in certain cells located in the proximal tubule of the cortex. Journal of Endocrinology (1993) 136, 85–89

Both sexual cycle and sleep circadian rhythms are disturbed by aging. It is known that these aging changes are involved in the functional disorder of the central nervous system. An attempt to clarify the central mechanism of the aging process in rats was made by comparing age‐related changes in the sleep circadian rhythm and sexual cycle with those in several centrally manipulated aging models. Old persistent estrous (PE with circadian PS: 9–14 months old) and 6‐OHDA treated rats known to show postsynaptic supersensitivity of s ‐adrenergic receptor showed the enhanced circadian rhythmicity in both slow wave (SWS) and paradoxical (PS) sleep, but PE with ultradian PS (11–24 months old) and intra‐suprachiasmatic (SCN) PCPA injected rats showed the circadian SWS and the ultradian PS rhythm. Old pseudopregnant (PSP) rats and rats bearing medial preoptic lesion indicated the circadian SWS and the semicircadian PS rhythm with higher night PS amounts. Old persistent diestrous (PD) and the rat bearing lar...

The effects of gonadectomy and gonadal hormone treatment of castrated rats or ovariectomized (OVX) rats bearing brain lesions on the circadian rhythms of slow wave sleep (SWS) and paradoxical sleep (PS) have been studied under a 14/10 light-dark schedule. Cortical EEGs and dorsal neck EMG were used to monitor SWS, PS and alertness. Intact female rats showed two daytime SWS peaks, one daytime PS peak and a small night PS peak except during proestrus. In intact male rats, the morning SWS peak and night PS peak were variable and SWS and PS peaks in daytime were dissociated. Orchidectomized (ORX) rats showed the morning SWS peak and disrupted the dissociation of SWS and PS peaks. Furthermore, gonadectomy increased the night PS peak. Posterior deafferentation of the hypothalamus (PDM) eliminated the night PS peak. Estradiol (E2B) injection to long term OVX rats eliminated the night PS peak from the first day of injection. However, E2B injection into androgenized OVX rats, ORX rats and OVX rats bearing septal lesion or MPO roof cut did not eliminate night PS peak. E2B injection to short term OVX rats or OVX rats with PDM lesions delayed the E2B-induced elimination of night PS peak. From these results, it is suggested that: (1) sexual dimorphism exists in the circadian sleep rhythm itself, and this difference partly depends on the hormonal environment produced by sex steroids; (2) the rise and fall of night PS peak reflects the neurohumoral environment in female rats; (3) the appearance of night PS peak involves the abolition of negative feedback of sex steroids and the posterior neural input into hypothalamus; and (4) the elimination of night PS peak on natural proestrus and following E2B treatment of OVX rats requires the intact positive feedback system of estradiol.

The effect of brain lesion or surgical isolation of the neural circuit on SWS and PS circadian rhythm have been studied in female rats under a 14/10 light-dark schedule. Cortical EEG'S AND DORSAL NECK EMG were used to monitor SWS, PS and alertness in female rats. Intact and operated controls showed regular 4-5-day vaginal cycles and nocturnal sleep rhythm, but the night PS value on proestrus was lower than in other cycles. Following septal lesion, MPO roof cut, vaginal cycles and SWS rhythm were regularly maintained; however, the PS appearance at night, except during proestrus, increased (night PS peak). These results were similar to those for pinealectomized or ovariectomized female rats. A frontal cut of the MBH produced persistent estrus and disturbed both SWS and PS circadian rhythm. The suprachiasmatic-lesioned rats showed persistent estrus and disrupted SWS rhythm, but regularly maintained the circadian PS rhythm. The vaginal cycles and SWS rhythm in the fornical-transected rats were regularly maintained, but the PS rhythm was disturbed during diestrus and showed ultradian rhythm. From these results, it is suggested that the pineal hormone and the gonadal feedback mechanisms may be involved in the night PS peak and this mechanism may involve the septal- and amygdaloid-hypothalamic systems. A different neural mechanism exist for SWS and PS circadian rhythm; SWS rhythm involves the suprachiasmatic-basal hypothalamic system and PS circadian rhythm is related, in part, to the hippocampal-hypothalamic system.

To examine the relationship between the sleep rhythm and the gonadal feedback system in the guinea pig, the effects of estrous cycle, gonadal steroids and brain deaf-ferentations on the sleep rhythm were studied and the following results were obtained;1) the guinea pigs did not show an apparent circadian rhythmicity in the sleep-wake-fulness cycle but showed an ultradian rhythm, whereas, the activity rhythm was circadian, 2) the rhythm in paradoxical sleep (PS) showed changes associated with the estrous cycle which were characterized by a decrease and rebound-like increase in PS amounts on the day of proestrus, 3) the horizontal deafferentation above the medial preoptic area at the level of the anterior commissure (MPO roof cut) did not disrupt the estrous cycle dependent changes in the PS rhythm, but the prechiasmatic deafferentation of the medial basal hypothalamus (PCD) and the large complete deafferentation of the medial basal hypothalamus (CDL) disrupted them, 4) ovariectomy (OVX) did not result in any changes in sleep and activity rhythms, 5) an administration of estradiol benzoate (E2) to OVX guinea pig caused a decrease in the amount of PS and an administration of progesterone (P) 48 h after E 2 caused a more pronounced decrease and rebound-like increase in the amount of PS, 6) the MPO roof cut did not affect the steroidal modification of the PS rhythm and the PCD disrupted it, while the CDL-animal also showed a E 2-induced PS decrease.From these results, it appears that the guinea pig may be a circadian animal, but this may not be seen in the sleep-wakefulness cycle, and the estrous cycle dependent changes in the PS rhythm may be the reflection of steroidal modification of the sleep rhythm and the site of action may be the inside of the medial preoptic anterior hypothalamic structures, but this area may also be affected by the output from the medial basal hypothalamus.