Evaluation of the pituitary-testicular function during experimental nephrosis.

To investigate the pituitary-testicular function in nephrotic rats, a sequence of experiments was undertaken in adult male rats after a single dose of puromycin aminonucleoside (PAN). Endocrine modifications were evaluated chronologically throughout the experimental disease in order to determine the appearance of hormone alterations which lead to the axis dysfunction. Serum concentration of LH, FSH, androstenedione, total and free testosterone, estradiol as well as urine testosterone were measured by specific RIAs on days 3, 7 and 10 after treatment on nephrotic and control groups. Prolactin was also evaluated on day 10. Likewise, total weight of various androgen responsive tissues from both groups was recorded, and the number of androgen receptor (AR) binding sites were determined. To know the functional status of the hipophyseal-testicular unit, groups of nephrotic and control rats were stimulated with LHRH (300 ng/100 g b.w.) or with one or four doses of hCG (8 UI), respectively. Additionally, the relative in vitro biological activity of FSH from nephrotic and control rats before and after LHRH stimulus was determined. The results from the hormonal profile revealed clear endocrine disorders characterized by a progressive diminution of all serum hormones except prolactin and urine testosterone, which remained unmodified. The weight of the main androgen responsive tissues, the ventral prostate and the seminal vesicle, decreased parallelly to androgen diminution. The binding analysis of AR shows a significant elevation of the available androgen sites in all analyzed tissues except kidney and hypothalamus. The secretion of LH and FSH from nephrotic animals after LHRH administration was lower than that from intact animals at the registered times. Interestingly, the biological activity of FSH from nephrotic rats was not detectable at both, before and after LHRH administration. Testicular response to hCG stimuli, in terms of testosterone synthesis was not significantly different in the two groups analyzed with respect to the intact animals. By contrast, no response was observed in terms of estradiol production at either one or four doses of hCG. On the whole, the results presented herein allow us to conclude that experimental nephrosis has a harmful effect on the pituitary-testicular axis, and strongly suggests that the endocrine dysfunction is initiated at the hypophyseal level; even though a specific testicular damage is also present.

Cerebral implants of histotypic hypothalamic cultures.

In the present report, the implantation of hypothalamic histotypic cultures within the cerebral hemisphere is described. The cultures were prepared from 10-day-old mice and rotary incubated during 6 or 10 days. When the cells reached their phenotypic characteristics at the end of the incubation period, the cultures were implanted in the posterior area of the lateral ventricle. 30 days postoperatively, the whole implanted area was removed and processed for light and electron microscopy observations. The implanted cultures completed maturation in the host tissue and no rejection signs were noticed; on the contrary, we were able to observe a favorable reaction from the host tissue, such as the formation of secondary blood vessels which penetrated the mass of the implanted culture. Well-developed magno- and parvocellular neurons were seen to contain neurosecretory vesicles in their terminals, and the neuroglial relationships established within these implants were homologable to those normally found.

Release of neurotransmitters and neurosecretory substances during in vitro maturation of mouse hypothalamic cultures.

The maturation of the neurosecretory activity of a hypothalamic nerve cell population grown in vitro, prepared from 10-day-old mice and cultured for 6 days, has been demonstrated in the present report. A low-molecular weight polypeptide of 30-kD was found to be released into the culture media during the 6-day period of incubation, as analyzed by SDS-polyacrylamide gel electrophoresis. Comparative electrophoresis of the in situ hypothalamic, neurohypophysis and cerebral cortex homogenates revealed the presence of a 30-kD protein component in both the hypothalamus and neurohypophysis but not in the cerebral cortex. The release of the 30-kD polypeptide into the incubation media indicates an expression of the neurosecretory activity of the peptidergic neurons of the hypothalamus during in vitro maturation. On the other hand, high pressure liquid chromatography with electrochemical detection showed appreciable quantities of released dopamine (DA), epinephrine and serotonin (5-HT) in the incubation media in which the neurons were allowed to differentiate. There was a steady release of DA during the 6-day incubation period, varying from 0.21 +/- 0.02 to 0.49 +/- 0.05 ng/mg protein. The epinephrine level increased progressively from day 1 to 6 of culture, from 3.73 +/- 0.57 to 12.08 +/- 1.81 ng/mg protein, respectively. The measured 5-HT level was 0.07 +/- 0.001 on day 2 and increased to 0.38 +/- 0.05 ng/mg protein on day 6 of culture. These data demonstrate the functional maturation of catecholaminergic, serotoninergic and peptidergic neurons in these rotary histotypic cultures of the mouse hypothalamus.

In vitro hypothalamic neurogenesis: morphological maturation of mouse hypothalamic cultures and in vitro versus in situ biochemical analysis.

The morphological and biochemical changes that occur during in vitro neurogenesis of the mouse hypothalamus were studied in rotary cultures prepared from mice between 4 and 16 days of postnatal age. After 6 days of in vitro growth, histotypic cultures with a high degree of morphological differentiation were obtained in cultures prepared from 8- to 10-day-old mice. Before day 8, the cultures showed immature neurons, while after day 12 most of them exhibited an undesired number of degenerated cells. Light-microscopic, Golgi and ultrastructural studies clearly showed the stages of development of the neurosecretory cells in culture. The particular organization of the hypothalamic cells in these cultures can be homologized to its equivalent region in vivo, as demonstrated by their morphological similarities as well as by the fact that the majority of the neurons orient their axons toward the external part of the culture in order to release the neurosecretory material outside the in vitro grown neuronal population, as is the case in situ since hypothalamic neurons release their neurosecretory products at the vascular system. Biochemical parameters such as DNA, RNA and protein contents were determined during the period of in situ development used for the preparation of histotypic cultures and compared to the biochemical changes that occurred during in vitro maturation. The changes in the in vitro DNA and protein contents showed the same variation pattern as in situ. The DNA/protein and RNA/protein ratios also had comparable characteristics, having peak values at days 10 and 16 in situ and in the histotypic cultures prepared from 10-day-old mice. These studies have demonstrated the correlation between the in vitro biochemical and morphological development and the significance of the critical period during hypothalamic neurogenesis for successful organotypic preparations.