Adrenocorticotropic hormone affects nonapoptotic cell death of undifferentiated germ cells in the fetal mouse testis: in vivo study by exo utero transplantation of corticotropic tumor cells into embryos.

Adrenocorticotropic hormone (ACTH) has been suggested to have possible roles in the fetal testes, one of the organs that express its specific receptors, melanocortin type 2 and 5 receptors (MC2R and MC5R), during the fetal period. We investigated the effect of ACTH on the cells in the testis cord of the fetal mouse testis by inducing ACTH-secreting AtT20 tumor cells in mouse fetuses. We first identified that mouse testicular germ cells at embryonic day (E) 16.5 and E18.5 spermatogonia were entirely CDH1 (E-cadherin)-positive by immunohistochemistry. We next performed AtT20-cell transplantation into the mouse fetus at E12.5, and analyzed ACTH effects on the development of fetal male mouse germ cells that express MC2R and MC5R at E16.5 and E18.5. The spermatogonia in the testis of AtT20-implanted embryos exhibited morphological changes, including pyknotic nuclei and swollen cytoplasm. In the AtT20-implanted embryos, the number of spermatogonia per unit area of the testis cord was significantly lower, but there were more pyknotic spermatogonia than in the controls. Single-stranded DNA-positive (apoptotic) and histone H3-positive (mitotic) spermatogonia were rarely observed and their numbers did not significantly differ in the two groups. Anti-Müllerian hormone (AMH)-positive Sertoli cells, another cell type that constitutes the fetal testis cord but does not express MC2R or MC5R, showed no apparent morphological changes compared with controls, nor were their numbers in the two groups significantly different between the two groups. These results suggest that ACTH, via MC2R and/or MC5R, may be involved in the nonapoptotic cell death of fetal mouse spermatogonia that is observed during the normal perinatal period.

Quantitative analyses of leukemia inhibitory factor in the cerebrospinal fluid in mouse embryos.

Leukemia inhibitory factor contributes to the self-renewal of neural stem cells in the forebrain. Although the existence of endogenous leukemia inhibitory factor in the brain parenchyma has been controversial, the cerebrospinal fluid is known to be another source of leukemia inhibitory factor. No reports of the measurement of leukemia inhibitory factor concentrations in the cerebrospinal fluid, however, exist. In the present study, we determined the leukemia inhibitory factor concentration in cerebrospinal fluid, amniotic fluid, and sera of embryos and dams in mice by enzyme-linked immunosorbent assay. The leukemia inhibitory factor concentrations were found to be constitutively high in the cerebrospinal fluid from embryonic day 11 to embryonic day 17, with a peak on embryonic day 13 and embryonic day 14. These findings correspond to the timing of cortical neuron production in mouse cerebrum.

Spatial and temporal patterns of expression of melanocortin type 2 and 5 receptors in the fetal mouse tissues and organs.

In the present study to analyze the role of ACTH in fetal tissues and organs, we observed the expression of melanocortin type 2 (MC2) and 5 (MC5) receptors in ICR mouse embryos from E11.5 to E18.5 by immunohistochemistry. In the adrenal gland and testis, both receptors were expressed from E13.5 to E18.5. In the genital ridge and the ovary, melanocortin type 2 receptors (MC2R) was detected from E11.5 to E12.5 and from E13.5 to E18.5, respectively, while melanocortin type 5 receptors (MC5R) was not detected. In the mesonephros, MC2R and MC5R were expressed from E11.5 to E12.5, and in the metanephros, MC2R and MC5R were expressed from E12.5 to E18.5 and from E14.5 to E18.5, respectively. In the lung, MC2R was expressed from E11.5 to E14.5, but MC5R was not expressed at all. In blood cells, MC5R was detected at all stages examined, while MC2R was detected at none. MC2R was observed in the brain and spinal cord from E11.5 to E13.5, while MC5R was detected only in the telencephalon and only from E16.5 to E18.5. At different temporal patterns, MC2R, but not MC5R, was detected in the choroid plexus, while MC5R, but not MC2R, was expressed in the liver and in the nasal epithelium, and both MC2R and MC5R were expressed in the dorsal root ganglion and the trigeminal ganglion. These findings show the spatio-temporal specific expression patterns of MC2R and MC5R in the mouse embryo and suggest that ACTH may be related to histogenesis and/or prenatal function of various tissues and organs via MC2R and/or MC5R.

Leptin deficiency causes pycnotic change in fetal cingulate cortical cells.

Leptin is an obese gene product, and leptin-deficient ob/ob mice develop hyperphagia and reduced locomotor activity. Leptin is thought to be related to brain development, because leptin receptors are widely expressed in the brain, and because brain weight as well as brain protein and DNA contents were reduced in adult ob/ob mice. In this study, we investigated the effect of leptin on the fetal cingulate cortex, since the leptin receptor is expressed in the neurons of the cingulate cortex, which is involved in emotion as well as in sensory, motor, and cognitive processes. The ob/ob fetuses had more pycnotic cells than wild-type fetuses in the cingulate cortex at embryonic day (E) 18. Many pycnotic cells were observed in the intermediate zone of the cingulate cortex. Most cells observed in this area were neuronal lineage cells, while few undifferentiated cells and oligodendrocyte precursor cells were found. At E18 there was no significant difference in the rostrocaudal length of the corpus callosum, which contains the neuronal projection from the cingulate cortex, between ob/ob and wild-type fetuses. We also showed that the length of the cerebrum was greater and the width of the cerebrum and cerebellum were lesser in ob/ob fetuses than in wild-type at E16. These results suggest an increased cell death in neuronal lineage cells in the intermediate zone of the cingulate cortex in leptin-deficient ob/ob mice. Leptin deficiency may also alter the gross morphology of the brain in development, but not the formation of the corpus callosum.

Leptin affects oligodendroglial development in the mouse embryonic cerebral cortex.

OBJECTIVE: Leptin, which is an obese gene product, decreases appetite and increases energy expenditure in adults. In our previous study, leptin was found to maintain neural stem cells and/or progenitor cells, preferentially astrocyte/oligodendrocyte progenitor cells, whereas it reduces the proportion of oligodendrocyte lineage-restricted precursor cells. It has been reported that leptin-deficient (ob/ob) mice have lower levels of glial proteins than wild-type mice. These findings suggest that leptin affects the development of glial cells. In this study, therefore, we investigated oligodendrocyte development in the cerebral cortex of ob/ob and wild-type mouse embryos by histochemistry. METHODS: We obtained ob/ob or wild-type (C57BL/6J) embryos on embryonic day (E) 18. We performed immunohistochemistry in the embryonic cerebrum with antibodies against NG2, platelet-derived growth factor receptor-alpha (PDGFR-alpha) and leptin receptor (Ob-R). In the cerebral cortex, we compared the number of the oligodendrocyte precursor cells (OPCs), which are immunopositive for NG2 and/or PDGFR-alpha, between ob/ob and wild-type embryos. RESULTS: We revealed that ob/ob embryos had significantly more OPCs than wild-type embryos on E18. PDGFR-alpha-positive OPCs did not coexpress leptin receptor in the cerebral cortex. CONCLUSION: These findings suggest that leptin inhibits differentiation of multipotent and/or glial progenitor cells into OPCs in the mouse embryonic cerebral cortex, but it does not directly act on OPCs.

Adrenocorticotropic tumor cells transplanted into mouse embryos affect pancreatic histogenesis.

A wide range of individual differences exist in the total number of functional and structural units in each organ, such as ß cells in pancreatic islands, and these units are the basis of the organ's overall function, including its functional reserve. The endocrine environment may influence organ histogenesis, during which functional and structural units are formed and increase in number. We analyzed the effects of a continuous high level of adrenocorticotropic hormone (ACTH) and/or secondarily induced glucocorticoid on histogenesis of the pancreas in mouse embryos. Pituitary tumor-derived AtT20 cells, which secrete ACTH continuously, were injected subcutaneously into mouse embryos at embryonic day (E) 12.5, and the embryos were allowed to develop exo utero until E18.5 (AtT20 group). E18.5 AtT20 group embryos with high ACTH levels (23.74 ± 6.19 ng/mL vs control group, 0.48 ± 0.40 ng/mL, P < 0.05) were examined for the effects on histogenesis of the pancreas. Using serial sections of the E18.5 pancreas, we stereologically measured the volumes, and counted total cell numbers and numbers of mitotic or pyknotic cells of the whole pancreas, endocrine and exocrine cells, and glucagon-immunopositive α cells and insulin-immunopositive ß cells in the endocrine part. Although the volumes of the whole pancreas and exocrine part did not change significantly, in the AtT20 group the endocrine part was significantly larger, with fewer pyknotic cells and lower ratios of α and ß cells than in the control group. These results suggest that the high level of ACTH and/or glucocorticoid affects histogenesis of the pancreas.

Individual variation in organ histogenesis as a causative factor in the developmental origins of health and disease: unnoticed congenital anomalies?

Morphological studies of congenital anomalies have mainly focused on abnormal shape (i.e. malformation) and thus on disturbed organogenesis. However, in regard to postnatal functions of organs that develop through branching mechanisms, organ size is another important morphological feature. These organs consist of a large number of structural and functional units, such as nephrons in the kidney, and the total number of these units, that is approximately proportional to the organ size, has been shown to vary widely among individuals. Organ-specific cells are differentiated and organized to form structural units and realize organ-specific functions during the histogenetic period (i.e. from mid-gestation to the early postnatal period). The total number of units is attained at the end of histogenesis and determines the total functional capacity, including the functional reserve of the organ, and thus may be related to predispositions to postnatal organ-based diseases, because the functional reserve decreases during the course of life and eventually become short of the minimum requirement of each organ. Therefore, it may be hypothesized that a smaller number of units of organs at the end of histogenesis is one of the predisposing factors for postnatal diseases (i.e. a form of unnoticed but late-manifested congenital anomalies), in this era of extended longevity. However, the mechanisms that control the total number of units in each organ during histogenesis and the possible relationship among the numbers of units in different organs remain unknown. Here, we review our trials based on the above hypothesis in order to (1) mathematically analyze the morphometric data of the different organs in fetuses to elucidate relationship among developing organs, (2) analyze the developing neuro-immuno-endocrine network as a series of mechanisms to systemically correlate the histogenesis of multiple organs, and (3) examine the maternal environment, including dietary fat, as a factor to influence histogenesis and thus the predisposition to type 1 diabetes.