Hypogonadism in the mutant hpg mouse is characterized by a deficiency of hypothalamic gonadotropin releasing hormone (GnRH). Affected male mice exhibit immature reproductive organs, small abdominal testes and low pituitary and plasma gonadotropin concentrations. Recent studies have demonstrated the potential of fetal brain transplants to establish functional connections with host tissues. We therefore sought to use this approach to correct the hpg deficit. Fetal preoptic area (POA) (a site of GnRH production) from unaffected animals of the hpg strain was transplanted into the anterior third ventricle of adult hpg mice. We report that in such implanted animals, killed 2 months post-implantation, the POA grafts contained GnRH neurones, from which GnRH-positive fibres could be traced to capillaries of the median eminence. Hypothalamic GnRH and pituitary and plasma gonadotropin concentrations were increased compared with levels in untreated (hpg) animals. The testes were enlarged and had descended into the scrotum. Evidence of full spermatogenesis and interstitial cell development was present in testicular sections. No such effects were seen with transplants of cortical tissue.

Gonadotropins-releasing hormone (Gn-RH) in selected regions of the female rat brain was measured by radioimmunoassay. Detectable immunoreactive Gn-RH was found in the anterior hypothalamic-septal region and in the mid-hypothalamic (arcuate-median eminence) region. Gn-RH was several times higher in the middle region than in the anterior region. Gn-RH was undetectable in the posterior hypothalamic region, frontal cerebral cortex and pineal glands, as well as in random blood samples, and low to undetectable in anterior pituitary glands. Gn-RH activity varied during the estrous cycle and after castration. In the mid-hypothalamic region, Gn-RH content was lowest throughout diestrus and in late morning and early afternoon of proestrus, and highest early in the morning of proestrus and during estrus. A significant decrease at mid-day was only found on the day of proestrus, a few hours prior to the critical period for LH release. In the anterior hypothalamic region, low Gn-RH activity was found from 1200 h of estrus to 1200 h of diestrus-2. A comparatively higher activity was seen at 1700 h of diestrus-2 and also from 1400 h of proestrus to 0800 h of estrus. Twenty-one days after ovariectomy, Gn-RH in the mid-hypothalamic region was significantly lower than the lowest values seen during the estrous cycle, while Gn-RH in the anterior hypothalamic region remained between low and high values seen during the cycle, being significantly higher than the low values. The changes observed during the estrous cycle and after castration suggest that gonadal steroids play a direct role in the control of hypothalamic Gn-RH. These data also demonstrate that Gn-RH varies in a different way in the anterior and mid-hypothalamic regions.

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Neural structures containing LHRH were characterized in the hypothalamus of the rhesus monkey by four different antisera to the hormone and an immunoperoxidase technique. Immunoreactive perikarya were present in a continuum from the septal-preoptic region anteriorly to the premammillary nucleus posteriorly. These cells were more concentrated in the pericommissural and tubero-infundibular regions. Reactive axons in the median eminence appeared to originate from the positive perikarya in the medial basal hypothalamus; this projection forms a tubero-infundibular tract containing LHRH. In addition, substantial numbers of fibers which entered the median eminence continued down the infundibular stalk and into the posterior pituitary. Other axons appeared to originate in the pericommisural region and projected to the organum vasculosum of the lamina terminalis. Scattered positive fibers were also present in other hypothalamic areas, especially in the periventricular zone and medical mammillary nucleus.

We found that two markers of differentiation, tyrosinase (monophenol, dihydroxyphenylalanine:oxygen oxidoreductase, EC 1.14.18.1) activity and melanin synthesis, are induced by diazepam in B16/C3 mouse melanoma cells. We also demonstrated high-affinity binding sites for [3H]diazepam in these cells by radioreceptor assay, and we visualized binding to the cell surface by fluorescence microscopy with a benzodiazepine analog conjugated to a fluorescein-labeled protein. Our studies also showed that there are differences between the binding characteristics in intact cells and in membrane fractions prepared from these cells. Scatchard analysis of the binding data from membrane fractions gave a linear plot (Kd = 9.1 X 10(-8) M). With intact cells, a curvilinear Scatchard plot was obtained. This was resolved into two components defining binding sites with affinity constants of 1.7 X 10(-9) M and 4.6 X 10(-7) M. Thus, it appears that [3H]diazepam binding in intact cells is more complex than in isolated membranes. Several related benzodiazepines, including flunitrazepam, Ro-5-4864, nitrazepam, oxazepam, lorazepam, Ro-5-3072, chlordiazepoxide, and clonazepam also induced melanogenesis. When these compounds were tested for their ability to inhibit [3H]diazepam binding, flunitrazepam, diazepam, and Ro-5-4864 were found to be the most effective inhibitors. These three compounds were also the most potent in inducing melanogenesis. Our results suggest that the benzodiazepines modulate cell differentiation. The presence of high-affinity binding sites in this homogeneous, easily grown cell line may provide a useful model for studies on the mechanism of action of these compounds.

A continuous cell culture line was established from a bone marrow metastasis of small cell anaplastic carcinoma of the lung. The cultures were characterized by light and electron microscopy, and an unusual concentric arrangement of cells was observed, both in sectioned material from the patient's tumor and from the cell cultures. The cells had two types of specialized cell junctions and contained secretory-like granules of the type described in neuroendocrine cells. Lactic dehydrogenase isozyme patterns were the same as those observed in normal human serum, and the karyotype revealed the presence of several marker chromosomes. Vasopressin was present in the cells and secreted into the culture medium in the absence of neurophysin, as shown by the immunoperoxidase technique and radioimmunoassay. Oxytocin was also absent from cells.

Experimental anatomical studies of the cortico-bulbar systems in several species of animals (guinea pig, Borri and Macchi, '60; rat, Valverde, '62; cat, Rossi and Brodal, '56; Kuypers, '58a; monkey and chimpanzee, Kuypers, '58b; and man, Kuypers, '58c) have indicated a basic %This investigation was supported (in part) by a research grant (NB 02540-03) from the National

The hpg mutant mouse lacks the neurohormone gonadotropin-releasing hormone (GnRH) and hence has a reproductive deficit. This deficit can be corrected by placement of normal fetal preoptic area into the third ventricle (see Krieger et al., 1985). We have now used ultrastructural immunocytochemistry to investigate the morphology of GnRH neurons in such intraventricular grafts, the routes that their axons take as they exit into the host, and the neurosecretory terminations that they make in the host median eminence. The GnRH cells in the transplant were similar in morphology to that reported for such cells in the preoptic area of other rodents. There was a large central nucleus, frequently indented and containing 1 or 2 nucleoli. The thin rim of cytoplasm was filled with rough endoplasmic reticulum, Golgi stacks, and mitochondria. Both dendritic and axonal profiles were identified, and a modest synaptic input to the former was found. Between the host and the implant a complex multilayered ependymal zone developed, and it was through this region that GnRH axons exited into the host arcuate nucleus and median eminence, usually surrounded by ependymal or glial elements. Within the median eminence, GnRH terminals were in close association with fenestrated blood vessels forming a normal neurosecretory terminus.