Motoharu Hayashi

Ablation of the scaffold protein JLP causes reduced fertility in male mice.

The specific and efficient activation of mitogen-activated protein kinase (MAPK) signaling modules is mediated, at least in part, by scaffold proteins. c-Jun NH2-terminal kinase (JNK)-associated leucine zipper protein (JLP) was identified as a scaffold protein for JNK and p38 MAPK signaling modules. JLP is expressed nearly ubiquitously and is involved in intracellular signaling pathways, such as the Gα13 and Cdo-mediated pathway, inxa0vitro. To date, however, JLP expression has not been analyzed in detail, nor are its physiological functions well understood. Here we investigated the expression of JLP in the mouse testis during development. Of the tissues examined, JLP was strongest in the testis, with the most intense staining in the elongated spermatids. Since the anti-JLP antibody used in this study can recognize both JLP and sperm-associated antigen 9 (SPAG9), a splice variant of JLP that has been studied extensively in primates, we also examined its expression in macaque testis samples. Our results indicated that in mouse and primate testis, the isoform expressed at the highest level was JLP, not SPAG9. We also investigated the function of JLP by disrupting the Jlp gene in mice, and found that the male homozygotes were subfertile. Taken together, these observations may suggest that JLP plays an important role in testis during development, especially in the production of functionally normal spermatozoa.

Comparative study of urinary reproductive hormones in great apes

Urinary estrone conjugates (E1C), pregnanediol-3-glucuronide (PdG), and follicle-stimulating hormone (FSH) were determined by enzyme immunoassays (EIAs) during the normal menstrual cycle in the orangutan, gorilla, chimpanzee, and bonobo. Furthermore, the data were compared to those levels in the human and long-tailed macaque. The results showed a typical preovulatory E1C surge and postovulatory increase in PdG in all species. The pattern of E1C during the menstrual cycle in the great apes more closely resembled the human than do the long-tailed macaque. A major difference of E1C pattern between these species appeared in the luteal phase. In the great apes and the human, E1C exhibited two peaks, the first peak detected at approximately mid cycle and the second peak detected during the luteal phase. On the other hand, in the long-tailed macaque, increase of E1C in the luteal phase was small or nonexistent. The gorilla, chimpanzee, and bonobo exhibited similar PdG trends. The orangutan excreted one tenth less PdG than these species during the luteal phase. The long-tailed macaque also excreted low levels of PdG. The patterns of FSH in orangutan, chimpanzee, bonobo and long-tailed macaque showed a marked mid-cycle rise and an early follicular phase rise, similar to those in the human. Comparing similar taxa, a large difference was found in FSH of gorilla; there were three peaks during the menstrual cycle. Thus, there is considerable species variation in the excretion of these hormones during the menstrual cycle and comparative studies could be approached with a single method. The methods and baseline data presented here provide the basis for a practical approach to evaluation and monitoring of ovarian events in the female great apes.

Differential expression of the truncated TrkB receptor, T1, in the primary motor and prefrontal cortices of the adult macaque monkey.

A truncated TrkB receptor, T1, which is one of the receptors for brain-derived neurotrophic factor, has been shown to regulate the morphology of neurons and glial cells in primary cultures and/or slices overexpressing T1 in the recent past. However, in vivo localization of T1 at protein level remains unclear. In the present study, we examined the localization of T1 in the primary motor and prefrontal cortices of adult monkeys by using immunohistochemistry. In the primary motor cortex, T1 immunoreactivity was observed mainly in the pyramidal neurons of layers II-VI, especially Betz cells of layer V. The apical and basal dendrites and cell bodies of Betz cells were strongly stained. In addition, we found that the interneurons were also T1-immunopositive and that there were no T1-positive astrocytes. In the prefrontal cortex, we observed strong immunoreactivity of T1 in astrocytes as well as pyramidal neurons of layer V. The pyramidal neurons and interneurons in layers II/III were faintly immunoreactive for T1. Thus, these findings, together with the fact that T1 is involved in morphological control of neurons and glial cells, suggest that the prefrontal cortex might possess a different degree of morphological plasticity than the primary motor cortex in the adult monkey.

Age-Related Changes of Elements in the Coronary Arteries of Monkeys in Comparison with Those of Humans

To elucidate compositional changes of the coronary artery with aging, the authors investigated age-related changes of elements in the coronary arteries of rhesus and Japanese monkeys by direct chemical analysis in comparison with the coronary arteries of Japanese and Thai. Used monkeys consisted of 38 rhesus monkeys and 23 Japanese monkeys, ranging in age from newborn to 33xa0years. After perfusion with a fixative, the hearts were resected from the monkeys, and the anterior interventricular branches of the left coronary artery and the right coronary arteries were resected from the hearts. After ashing of the arteries, element contents were determined by inductively coupled plasma-atomic emission spectrometry. It was found that the Ca and P contents did not increase in both the left and right coronary arteries of rhesus and Japanese monkeys at old age. The average contents of Ca and P decreased by 13% and 25% in the left coronary arteries more than 20xa0years of age in comparison with those below 20xa0years of age, whereas they decreased by 4% and 15% in the right coronary arteries more than 20xa0years of age in comparison with those below 20xa0years of age. This finding indicated that atherosclerosis scarcely occurred in both the left and right coronary arteries of rhesus and Japanese monkeys at old age. In contrast with monkeys, atherosclerosis occurred frequently in the coronary arteries of Japanese and Thai at old age.

Development of full-length Trk B-immunoreactive structures in the prefrontal and visual cortices of the macaque monkey

Distribution and morphological changes of cells containing the signal transducing neurotrophin receptor, full-length Trk B (fl-Trk B), were investigated in the prefrontal cortex (area FD) and the primary visual cortex (area OC) of the macaque monkey between embryonic day 140 and the adult stage. In area FD at the adult stage, fl-Trk B immunoreactivity was mainly observed in the pyramidal cells in layers II/III, V and VI. Small numbers of granule cells in layer IV were immunopositive. Bipolar and multipolar cells in layer II were rarely immunoreactive. At embryonic day 140, the number of fl-Trk B immunoreactive pyramidal cell was high, and gradually decreased until the adult stage. In layer IV, the number of fl-Trk B-ir cells was also high at embryonic day 140, and decreased remarkably from postnatal day 7 to the adult stage. On the other hand, in area OC at the adult stage, cells in layers II/III, IV, V and VI were fl-Trk B immunopositive. From embryonic day 140 until adulthood, the cells in layer IVc were fl-Trk B immunoreactive. The strongest fl-Trk B immunoreactivity in areas FD and OC occurred at postnatal month 6, coinciding with the time of the synapse overproduction. These findings suggest that ligands of fl-Trk B, such as BDNF and NT4/5 may be involved in the development and maintenance of the monkey cerebral cortices.

Development of full-length Trk B-immunoreactive structures in the hippocampal formation of the macaque monkey.

u2002Distribution and morphological changes of cells containing the signal transducing neurotrophin receptor, full-length Trk B (fl-Trk B), were investigated in the hippocampal formation of the macaque monkey between embryonic day 140 and the adult stage. Western blot analysis showed that one main protein band, which migrated at 141xa0kDa, was detected in both the embryonic and adult hippocampal formation. In the pyramidal cells in CA1 and CA3 subfields, the subiculum, and the entorhinal cortex, fl-Trk B-immunoreactive dendrites were observable in the embryonic stage. In contrast, in the granule cells of the dentate gyrus, few dendrites were immunoreactive during embryonic and early developmental stages. This difference may be due to the later growth of the granule cells of the dentate gyrus. The existence of fl-Trk B immunoreactivity in the cell body and dendrites in the embryonic hippocampal neurons, suggests that BDNF and/or NT4/5 act on the hippocampal cells by autocrine/paracrine mechanisms. In the entorhinal cortex, fl-Trk B immunoreactivity became localized in the stellate cells in layer II and the pyramidal cells in layers III,V and VI in adulthood. This indicates that BDNF and/or NT4/5 are important for the maintenance of the projection neurons in the entorhinal cortex at the adult stage. The strongest fl-Trk B immunoreactivity in the hippocampal neurons occurred at postnatal month 4, corresponding to the period of greatest synapse production in the monkey hippocampus, suggesting that BDNF and/or NT4/5 with fl-Trk B may play a role in synapse formation in the monkey hippocampus.

Developmental changes in the expression of growth-associated protein-43 mRNA in the monkey thalamus: Northern blot and in situ hybridization studies

The expression of growth-associated protein-43 has been related to axonal elongation and synaptic sprouting. Using the Northern blot analysis, we investigated the developmental changes of growth-associated protein-43 mRNA in the thalamus of macaque monkeys. The amount of growth-associated protein-43 mRNA was high at embryonic day 125, and decreased at postnatal day 1. It increased again at postnatal day 8, reached its peak value at postnatal days 50-70, and then decreased gradually until postnatal year 1. We previously reported that the amount of growth-associated protein-43 mRNA in the cerebral cortex decreased roughly exponentially during perinatal and postnatal periods and that it approached the asymptote by postnatal day 70 [Oishi T, Higo N, Umino Y, Matsuda K, Hayashi M (1998) Development of GAP-43 mRNA in the macaque cerebral cortex. Dev Brain Res 109:87-97]. The present findings may indicate that extensive synaptic growth of thalamic neurons continues even after that of cortical neurons has finished. We then performed in situ hybridization to investigate whether the expression level of growth-associated protein-43 mRNA was different among various thalamic nuclei. In the infant thalamus (postnatal days 70-90), moderate to intense expression of growth-associated protein-43 mRNA was detected in all thalamic nuclei. Quantitative analysis in the infant thalamus indicated that the expression levels were different between the nuclear groups that are defined by the origin of their afferents. The expression in the first order nuclei, which receive their primary afferent fibers from ascending pathways [Guillery RW (1995) Anatomical evidence concerning the role of the thalamus in corticocortical communication: a brief review. J Anat 187 (Pt 3):583-592], was significantly higher than that in the higher order nuclei. While moderate expression was also detected in the adult dorsal thalamus, the expression in the first order nuclei was almost the same as that in the higher order nuclei. Thus, the in situ hybridization experiments indicated that the transient postnatal increase in the amount of growth-associated protein-43 mRNA, which was shown by the Northern blot analysis, was mainly attributed to enhanced expression in the first order nuclei during the postnatal period. This may be a molecular basis for environmentally induced modification of thalamocortical synapses.

Levels of serum brain-derived neurotrophic factor in primates

Brain-derived neurotrophic factor (BDNF) has been reported to exist not only in nervous tissue but also in serum. In contrast to the wealth of knowledge regarding the various physiological functions of BDNF in the nervous system, information about possible roles in other systems is limited. To elucidate the physiological function of serum BDNF in primates, it is first necessary to establish a method to determine the levels of BDNF in serum of primates. In the present study, we established an enzyme-linked immunosorbent assay (ELISA) method which we used to measure levels of serum BDNF in non-human primates. We found that serum BDNF levels were similar among several species of primates. The present results suggest that our BDNF ELISA may be useful in measuring serum BDNF concentration as a physiological marker, and that levels of serum BDNF may be similar among primates including humans.

Age-related attenuation in the elements in monkey sino-atrial node

Changes in trace elements of the sino-atrial (SA) node with aging was investigated using 24 hearts of the Japanese and rhesus monkeys of ages ranging from 27 d to 30 yr. With aging, sympathetic activity decreases and SA nodal function deterionates. The SA nodal tissue was removed from the anatomical position and was confirmed by means of histological observation. The elements, such as Ca, P, S, Mg, Na, Fe, and Zn, were analyzed using inductively coupled plasma-atomic emission spectrometry (ICP-AES). Advancing age never increased the contents of the trace elements, but decreased them. The correlation coefficients for the age-dependent attenuations were −0.561 (n=24, p<0.01) in Ca and −0.482 (n=24, p<0.05) in P. The correlations for the attenuations induced by other trace elements were not significant. Furthermore, close relationships of the elements between Ca and P, S, Zn, or Na contents, between P and Zn or Na contents, and between Zn and Na contents were observed. These results indicate that the elements in the monkey SA node are attenuated with an increase in age, presumably suggesting the age-related suppression of cardiac functions as a result of the histological alterations of the SA nodal cells.

Inhibin B is the major form of inhibin secreted from testes in male Japanese macaques (Macaca fuscata)

In order to clarify the cellular source and forms of bioactive inhibin in male Japanese macaques (Macaca fuscata), circulating concentrations of inhibin A and B, and immunohistochemical localization of inhibin subunits in testis were studied. Plasma concentrations of testosterone were also measured. The present study showed that inhibin B was clearly detected in the plasma of male Japanese macaques. Moreover, concentrations of both inhibin B and testosterone during the breeding (mating) season were significantly higher than those of the non-breeding season. On the other hand, plasma inhibin A was detected neither during the breeding seasons nor during the non-breeding seasons. Positive stainings with α and βB subunit antibodies were observed in the Sertoli cells, however staining with βA subunit antibody was not observed in the testicular samples. These results indicate that inhibin B is the major circulating inhibin and probably secreting from Sertoli cells in male Japanese macaques.