Shizuko Murakami

Estrogen and apoptosis in the developing sexually dimorphic preoptic area in female rats

Effect of estrogen on apoptotic cell death was studied in the two sexually dimorphic neuronal groups in the developing rat preoptic area (POA): the anteroventral periventricular nucleus of the POA (AVPvN-POA); and the sexually dimorphic nucleus of the POA (SDN-POA). A specific labelling of nuclear fragmentation was performed by terminal deoxynucleotydyl transferase(TdT)-mediated dUTP-biotin nick end-labeling method (TUNEL method) to demonstrate apoptosis. In the AVPvN-POA whose size is larger in females than in males, the number of TUNEL-positive cells was not significantly different between day 5 control and female pups sacrificed 10 h after 25 micrograms estradiol benzoate (EB) injection. However, TUNEL-positive cells showed a significant increase in the female pups sacrificed 24 h after EB injection, compared to that shown in the control female pups. In the SDN-POA whose size is larger in males than in females, EB injection significantly decreased TUNEL-positive cells in the female pups sacrificed 24 h after EB injection, compared to that in controls. These results suggest that estrogen regulates the neuronal number by facilitating apoptotic cell death in the developing AVPvN-POA or by inhibiting it in the developing SDN-POA.

The ontogeny of luteinizing hormone-releasing hormone (LHRH) producing neurons in the chick embryo : Possible evidence for migrating LHRH neurons from the olfactory epithelium expressing a highly polysialylated neural cell adhesion molecule

The development of neurons expressing luteinizing hormone-releasing hormone (LHRH) has been studied immunohistochemically in the chick embryo from the 3.5 embryonic day (ED) to the day of hatching. At ED-3.5, LHRH-immunoreactive neurons were first detected in the medial epithelium of the olfactory pit, but their appearance in the brain was delayed to ED-4.5. On EDs-6-7, cords of the LHRH-immunoreactive cells extended across the nasal septum towards the ventromedial forebrain with the olfactory nerve. By double staining for LHRH and, a highly polysialylated form of neural cell adhesion molecule (NCAM-H), the LHRH-positive neurons in the olfactory-forebrain system were found strongly NCAM-H-positive. At ED-8, a marked decrease in the number of LHRH-positive cells in the olfactory epithelium and a concomitant increase in the LHRH-positive cells in the forebrain area were noted. From ED-11 to the day of hatching, the majority of LHRH-positive neurons tended to move into their usual adult position, whereas the LHRH-positive cells had almost disappeared in the olfactory epithelium. No LHRH-immunoreactive neurons were found strongly positive to NCAM-H. These results suggest that LHRH neurons originate from the olfactory placode, then as they develop they migrate across the nasal septum and enter the forebrain with the olfactory nerve. The close association of NCAM-H with the developing LHRH neurons raises the possibility that NCAM-H plays some role in guiding the migrating LHRH neurons from the olfactory epithelium to the forebrain.

The origin of the luteinizing hormone-releasing hormone (LHRH) neurons in newts (Cynops pyrrhogaster): the effect of olfactory placode ablation

SummaryNeurons containing luteinizing hormone-releasing hormone (LHRH) are first detected in newt embryos (Cynops pyrrhogaster) in the olfactory epithelium and ventromedial portion of the olfactory nerve, after which they sequentially appear in the intracerebral course of the terminal nerve at prometamorphosis, and in the septo-preoptic area at postmetamorphosis. In adults, however, LHRH-immunoreactive cells are rarely seen in the nasal region, and their distribution shifts into the brain, suggesting their migration. In order to ascertain the origin and possible migration route of these neurons in newt larvae, the effect of unilateral or bilateral olfactory placodectomy on the LHRH neuronal system has been studied. Removal of the olfactory placode results in the absence of LHRH-immunoreactive cells in the nasal and brain regions of the operated side, whereas the subsequent growth and the LHRH-immunoreactive cellular distribution in the contralateral side are identical to those of normal larvae. Following bilateral placodectomy, no LHRH immunoreactivity is detected on either side of the olfactory-brain axis. These results suggest that LHRH neurons of the newt, Cynops pyrrhogaster, originate in the olfactory placode and then migrate into the brain during embryonic development.

Neuronal death in the developing sexually dimorphic periventricular nucleus of the preoptic area in the female rat: Effect of neonatal androgen treatment

Neonatal treatment of female rats with androgen decreases the nuclear volume of the anteroventral periventricular nucleus of the preoptic area (AVPv-POA). In order to examine the effect of androgen on the neural substrates in the developing AVPv-POA which show a sexual dimorphism in nuclear volume, the cell death pattern in the AVPv-POA was compared between normal females and androgenized females. Wistar female rats were treated with 50 micrograms of testosterone propionate (TP) for 5 days from the day of birth. Degenerating cells (pyenotic cells) and normal cells were counted in every third section from days 1 to 13 of life. The rate of pycnotic cells to 1000 cells in TP-treated females sacrificed at days 7 and 10 was significantly higher than that in normal females. These results may suggest that neonatal androgen regulates neuronal death in the AVPv-POA, decreasing the number of neurons in the nucleus.

Androgen Enhances Neuronal Degeneration in the Developing Preoptic Area: Apoptosis in the Anteroventral Periventricular Nucleus (AVPvN-POA)

Perinatal treatment of female rats with androgen decreases the nuclear volume of the anteroventral periventricular nucleus of the preoptic area (AVPvN-POA). In order to examine the effect of androgen on neurogenesis, bromodeoxyuridine (BrdU) was given once on Day 15 of gestation (= E15) to pregnant rats that also received testosterone propionate (TP) injections. When examined at E17, the number of BrdU-labeled neurons in the AVPvN-POA was not significantly different among control female, male, and androgenized female fetuses, suggesting that androgen does not interfere with neurogenesis. At E21, a significant reduction of BrdU-labeled AVPvN neurons was observed in males and androgenized females. These findings support the hypothesis that elimination of a population by cell death is enhanced in males and androgenized females. Similar selective elimination of the AVPvN neurons occurred in the female following neonatal TP treatment. In order to investigate the nature of androgen-induced cell death in the AVPvN-POA, specific labeling of nuclear DNA fragmentation was performed by the TdT-mediated dUTP-biotin nick end-labeling (TUNEL) method. The number of TUNEL-positive cells was significantly greater in neonatally androgenized females, compared to that in control females. Since DNA fragmentation is considered the most characteristic feature of apoptosis, and TUNEL method is based on direct, specific labeling of DNA fragmentation in nuclei in situ, the neuronal death in the AVPvN-POA is apoptotic, and perinatal androgen may induce the selective apoptotic cell death in the AVPvN-POA.

Origin of luteinizing hormone-releasing hormone (LHRH) neurons in the chick embryo: effect of the olfactory placode ablation.

A unilateral olfactory placodectomy resulted in the absence of luteinizing hormone-releasing hormone-immunoreactive (LHRH-ir) cells in the olfactory-forebrain axis of the operated side, whereas the development of the LHRH neuronal system was not disturbed on the unoperated side. In the embryos in which a fragment of the medial olfactory epithelium was spared the damage, a small number of LHRH-ir cells were detected in the nasal region of the operated side, where the lack of the central projection of the olfactory nerve caused stagnation of LHRH-ir cells, no ir-cells being found in the brain area. These results suggest that LHRH neurons originate in the olfactory placode and then migrate into the forebrain along the olfactory nerve.

Enzymatic removal of polysialic acid from neural cell adhesion molecule perturbs the migration route of luteinizing hormone-releasing hormone neurons in the developing chick forebrain.

During development in the chick embryo, luteinizing hormone‐releasing hormone (LHRH) neurons migrate along the olfactory nerve from the olfactory epithelium to the forebrain. At embryonic day 5.5 (E5.5) to E6.0, the majority of LHRH neurons begin to enter the medial forebrain and then course dorsocaudally along the forebrain substance just beneath the pia matter in association with the somatostatin (SST)‐positive fibers, which branch medially from the SST‐positive olfactory nerve. By E6.5, the neurons and SST‐positive medial branch of the olfactory nerve have proceeded toward the septal area. Intense immunoreactivity for the polysialylated form of neural cell adhesion molecule (PSA‐NCAM) on both the LHRH neurons and the SST‐positive fibers during this period suggests that this less adhesive form of NCAM is involved in the migratory process. This possibility was examined by using a polysialic acid (PSA)‐specific endoneuraminidase. PSA removal did not alter the behavior or appearance of the SST‐positive olfactory fibers within the migration pathway. However, it induced a significant deviation of migrating LHRH neurons from the regular path in the forebrain. The effect of PSA removal is more likely to involve changes in the interaction of the migrating neurons with a subset of the SST‐positive olfactory fibers and/or other elements in the forebrain rather than an alteration in the pattern of their axonal substrate. On the basis of these results, it is suggested that PSA contributes to the specific pattern of LHRH neuronal migration in the forebrain by limiting interaction of these LHRH neurons with their surrounding environment. J. Comp. Neurol. 420:171–181, 2000.

Transient expression of somatostatin immunnoreactivity in the olfactory-forebrain region in the chick embryo

The tissue distribution of somatostatin (SST) immunoreactivity was studied in the nasal and forebrain region in the chick embryo. On embryonic day (ED) 3, SST-immunoreactive (ir) cells were first detected in the cells migrating from the olfactory placode. Then, at ED3.5, SST-ir cells and -ir fibers appeared in the olfactory epithelium and olfactory nerve bundles. At ED6-8, one component of the SST-ir fibers was found to separate from the olfactory nerve and it entered the parenchyma of the medial forebrain surface. These SST-ir fibers extended dorsocaudally toward the preseptal area. During this same period, a few SST-ir cells were observed in the medial forebrain adjacent to the SST-ir fibers. SST immunoreactivity in the nasal and forebrain areas was most striking at ED5-8 but a reduction of SST immunoreactivity in the nasal and forebrain areas occurred at ED11 and it virtually disappeared by the day of hatching. These results indicate that the expression of SST in the nasal and forebrain regions is transient in the chick embryo. Since the SST-ir cells did not co-express luteinizing hormone-releasing hormone (LHRH), it, thus, appears that these SST-r cells belong to a different cell population from LHRH neurons that are also found in the olfactory-forebrain axis during embryonic development [23]. However, a close relationship exists between SST-ir cells and -ir neuronal fibers and LHRH neurons. This may play a role in development of LHRH neurons.

Direct evidence for the migration of LHRH neurons from the nasal region to the forebrain in the chick embryo: a carbocyanine dye analysis

To examine the possible migration of luteinizing hormone-releasing hormone (LHRH) neurons from the olfactory placode to the brain, the epithelial cells of the olfactory placode of the chick embryos were labeled with a fluorescent carbocyanine dye DiI at embryonic days 3.5-4.0. The embryos treated with DiI were incubated in ovo for 1 to 7 days. The DiI-labeled cells were first detected in the olfactory epithelium and olfactory nerve one day after the application of DiI. Two to four days after the application of the dye, the labeled cells sequentially appeared in the rostral and medial forebrain, and in the septo-preoptic area. The distribution pattern of DiI-labeled cells closely resembled that of LHRH neurons. Double staining for DiI and LHRH demonstrated that DiI-labeled cells co-expressed LHRH. However, there were a number of DiI-labeled cells which did not co-express LHRH. These results provide evidence for the actual migration of LHRH neurons from the olfactory placode to the septo-preoptic area during the development, and also suggest that some non-LHRH neurons in the forebrain are of olfactory placodal origin.

Straying phenomenon of migrating LHRH neurons and highly polysialylated NCAM in the chick embryo

The present study on unilaterally incompletely placodectomized chick embryos revealed that arrival of migrating LHRH neurons into the forebrain area is dependent on the presence of a central projection of the olfactory nerve. When a fragment of the lateral and medial olfactory epithelium was spared the damage, a small number of LHRH-ir cells were found to migrate into the forebrain along a thin NCAM-H-expressing fiber bundle of the olfactory nerve. In embryos with large lesions which destroyed the lateral olfactory epithelium, the poorly developed NCAM-H-positive olfactory nerve fibers were arrested where they meet the NCAM-positive medial nasal branch of the ophthalmic nerve and frequently fused with this branch fiber bundle. In these embryos, no LHRH-ir cells were detected in the forebrain area. They were deviated from their regular migratory course to the NCAM-H-positive medial nasal branch fiber bundle. This straying phenomenon indicates that some structural support is apparently needed for the migration of LHRH-ir cells, but their migratory route is not completely programmed in their precursor cells in the olfactory placode. In cultures of placodal tissues, the coexistence of migrating LHRH-ir cells with NCAM-H-expressing neural elements was always confirmed. It is suggested therefore that not only the structural support for the migration but also the interaction between LHRH-ir cells and NCAM-H-expressing neural elements is a prerequisite for successful LHRH neuronal migration.