Biomedical subjects
Publications and source records attributed to W B Neaves.
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In the current investigation, subcellular particles (synaptosomes) of hypothalamic homogenates were isolated by differential centrifugation and discontinuous sucrose density gradient fractionation and found to be rich in LHRH, TRH, and the neuronal marker, norepinephrine (NE). Of the total quantity of LHRH, TRH, or NE in the nuclei-free homogenate, 52-65% was recovered in synaptosomes, whereas the cytosol, myelin/microsomes, and mitochondria contained only 1-12%. To determine the subsynaptosomal localization of LHRH and TRH, purified synaptosomes were lysed and the resulting suspensions were fractionated on discontinuous sucrose density gradients. LHRH (30-40%) was found to be localized primarily in subsynaptosomal particles which banded at sucrose densities between 0.6-1.0 M. Electron micorscopic analysis of these particles revealed the presence of dense-cored granules (70-80 nm diameter) and synaptosomal membrane remnants. Norepinephrine was found in two pools within the isolated nerve endings: 15-25% of synaptosomal NE was associated with the synaptic vesicles (45-55-nm diameter); about 40% was in the cytosol. TRH was present primarily as a soluble component of the nerve ending. No apparent association of TRH with dense-cored granules was demonstrable in this study; however, there may be some TRH in synaptic vesicles.
By 3 months after surgery, testicular weights were 12-14% below control values in a large series of vasectomized rats of the inbred Lewis strain. Testicular sperm concentrations in the vasectomized rats were 25-29% below those in the sham-operated animals. Although modest in magnitude, these differences were highly significant (P less than 0.001). Histometric analysis of testes from a smaller series of vasectomized Lewis rats revealed various proportions of abnormal seminiferous tubules. The degenerative changes included reduction in tubule diameter, fusion of spermatids, and depletion of advanced germinal elements. In the most severely affected tubules, leakiness of the blood-testis barrier was indicated by penetration of lanthanum through Sertoli-cell tight junctions.
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The 900 x g supernatant fluid prepared from hypothalamic homogenates from male and female rats (ranging in age from -1 to 120 days) was fractionated by means of continuous sucrose density gradient centrifugation. Thyrotropin releasing hormone (TRH) and LH releasing hormone (LHRH) in the gradient fractions were quantified by radioimmunoassay. In adult hypothalamic homogenates, TRH and LHRH were associated with two populations of particles distinguishable by their sedimentation properties. Each peptide was in turn distributed in two subpopulations of parties differing in size but similar in density. The distribution of each peptide within its subpopulations of particles was found to be a function of age. In hypothalami of 22-day-old fetuses, TRH was associated almost entirely with the subpopulation of small particles. However, in the neonates, an age-dependent increase in the fractional amount of the TRH confined to the subpopulation of large particles was observed. By the 7th day of age, the peptide was equally distributed in the two subpopulations. The buoyant density of the 1-day-old neonatal particles and that of the adult small and large particles containing TRH was similar. The ontogeny of the subcellular compartmentalization of LHRH differed appreciably from that of TRH. LHRH was barely detectable in hypothalami of 22-day-old neonates. Nevertheless, at this age, the peptide was confined primarily to the subpopulation of large particles, and a similar compartmentalization was noted in hypothalami of 5- and 7-day-old neonates. However, in hypothalami of 14-day-old males and 21-day-old females, association of LHRH with the subpopulation of small particles was evident. It is concluded that 1) the nature of the hypothalamic subcellular compartmentalization of TRH and LHRH is age dependent, 2) the compartmentalization of each peptide in neonatal hypothalami differs from that in the adults, and 3) the development of the mature profile of subcellular compartmentalization of TRH and LHRH proceeds asynchronously.
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During the annual breeding season, the testes of the lizard Cnemidophorus gularis are yellow-orange, oviod organs measuring almost 1 cm in greatest diameter. The pigment is confined to the testicular tunic, which contains a zone of Leydig cells and vascular channels more than 50 mu thick. Leydig cells constitute approximately 60% of the zone, with remaining space occupied by capillaries, sinusoids, and lymphatic vessels. Lymphatics are concentrated at the interface between tunic and seminiferous tubules. Interstitial space is poorly developed among the tubules, accounting for less than 3% of tissue volume. Capillaries, lymphatics, and few widely scattering Leydig cells occur in the sparse interstitial space. Leydig cells in the tunic and elsewhere in the testis show ultrastructural features commonly found in mammalian Leydig cells. Separation of the tunic from the seminiferous tubules is achieved in a few seconds by manual decapsulation of the testis and yields an enriched preparation of Leydig cells that is essentially uncontaminated by tubular elements.
C57BL/6J mice inoculated sc with 50 mg of a transplantable Leydig cell tumor (M5480) demonstrated a reproducible pattern of slow tumor growth up to day 10 followed by rapid growth at the rate of approximately 0.5 g per day through day 27. The mean survival time of tumor-bearing mice was 33 days, when tumor weight accounted for more than 25% of total body weight. The rapid increase in weight occurring around day 10 resulted largely from a 20-fold increase in the quantity of extravasated blood inside the tumor, which in turn promoted a 50% reduction in host hematocrit. Sustained enlargement of M5480 during the third and fourth weeks of growth was supported by proliferation of tumor cells. Apart from blood-filled cavities, over 90% of the tumor consisted of neoplastic Leydig cells exhibiting generalized cytoplasmic features usually associated with mitotic activity. An activated macrophage was the next most abundant cell type, accounting for 2-3% of the nucleated cell mass. The remaining 3% was occupied by vascular elements, leukocytes, and giant cells. Depending on the age of the tumor, varying proportions of the cell population showed signs of anoxic degeneration. Degenerate cells were minimal at day 14, accounting for less than 4% of the total population, and maximal beyond day 21, when they occupied more than 50% of the cell mass.
Several factors related to androgen status were examined in vasectomized and sham-operated rats during and at the end of the 7th postoperative month. Average serum testosterone levels during the 7th month were based on three biweekly bleedings of 12 rats in each group, with determined values of 1.84 plus or minus 0.61 ng/ml (X plus or minus SD) for vasectomized rats and 2.38 plus or minus 0.78 ng/ml for sham-operated rats. Although average serum testosterone levels were 23% lower in vasectomized rats as compared to sham-operated controls, high coefficients of variation (33% and 32% in the two groups, respectively) rendered the difference insignificant. Within 1 hr of hCG administration, serum testosterone rose to 5.07 plus 1.94 ng/ml and 5.73 plus or minus 3.21 ng/ml in vasectomized and sham-operated rats. Eighteen hr after castration, testosterone had fallen below 0.25 ng/ml in both groups. Weights of testes and accessory sex organs were similar in both groups. Significant differences were not detected in the abundance, volume, or ultrastructure of Leydig cells. Each category of data collected in this study failed to reveal statistically significant differences were not detected in the abundance, volume, or ultrastructure of Leydig cells. Each category of data collected in this study failed to reveal statistically significant differences between the two groups, thereby supporting the essential normalcy of vasectomized rats with respect to androgen status.
The development of gonadotropin receptors for LH and hCG in the fetal rabbit testis from 17-29 days of gestation was followed by quantitative binding studies with [125I]iodohCG and compared with gonadal testosterone content and the histological differentiation of the fetal Leydig cells. The concentrations of gonadotropin receptors and testosterone in the fetal testis were low on days 17 and 18 and increased strikingly on day 19. This time sequence for development of LH-hCG receptors and steroid content of the testis was correlated exactly with the histological appearance of the endoplasmic reticulum characteristic of the differentiated Leydig cell. When fetal testes were examined at 12-h intervals between days 17 and 19, gonadotropin binding and testosterone content were closely correlated at all times studied. Thus, no dissociation between the two functions was demonstrable in the testis at any time during gestation. In the fetal ovary, LH-hCG binding and testosterone content were low or undetectable at all stages of gestation. These observations demonstrate a close temporal relationship between the appearance of the LH-hCG receptor and the synthesis of testosterone by the fetal testis and demonstrate that the histological and functional differentiation of the Leydig cell occurs within a few hours at approximately day 18 of gestation. The simultaneous appearance of LH-hCG receptors and testosterone synthesis in the gonad can be regarded as the biochemical manifestations of Leydig cell differentiation in the testis of the fetal rabbit.
The development of gonadotropin-induced structural change in a testosterone-secreting Leydig cell tumor and its relationship to plasma testosterone levels were studied in castrate, tumor-bearing mice 2 hr after daily injections of human chorionic gonadotropin (HCG). Tumor cells from control animals were small, averaging less than 1.0 X 10(-9) ml in volume, and were poorly differentiated, having very little smooth endoplasmic reticulum (SER) in their cytoplasm. Average plasma testosterone levels in these mice were near 1.3 ng/ml. Although a five-fold rise in plasma testosterone was measured 2 hr after the first HCG injection, no changes in the endoplasmic reticulum were detected at this time. After the second injection, plasma testosterone rose to only twice control levels, while ultra-areas of mixed rough endoplasmic reticulum and SER. Modest amounts of SER had appeared in many tumor cells after the third injection, and plasma testosterone showed a six-fold rise. Between the third and fourth injections, cell volume increased by about 70% as large accumulations of SER appeared in the cytoplasm of most cells. Plasma testosterone again rose to five times control levels. Increased cell volume and abundant SER were maintained by continuing daily injections of HCG, while the 2-hr plasma testosterone response persisted at five to six times the control level. These findings show that a maximal elevation of plasma testosterone can occur prior to changes in the endoplasmic reticulum of Leydig tumor cells. However, they also suggest that daily repetition of the maximal functional response requires that the cells acquire large quantities of newly produced SER.
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The permeability of Sertoli cell tight junctions to lanthanum administered during fixation has been compared in rats after ligation of the ductus deferens and after ligation of the ductuli efferentes. In both control and vasoligated testes, lanthanum penetrated only short distances into the Sertoli cell tight junctions before stopping abruptly. The tight junction, consisting of numerous pentalaminar fusions of contiguous Sertoli cell membranes, prevented diffusion of lanthanum into the adluminal compartment of the seminiferous epithelium. In rats with ligated ductuli efferentes, lanthanum completely permeated many Sertoli cell tight junctions and occupied intercellular spaces of the adluminal compartment. In spite of their newly acquired permeability to lanthanum, tight junctions retained characteristic ultrastructural features, including numerous membrane fusions. When lanthanum-filled tight junctions were sectioned en face, membrane fusions appeared as pale lines in lakes of electron-opaque tracer. These linearly extensive fasciae occludentes occasionally ended blindly, suggesting that lanthanum may have traversed the junction by diffusing around such incomplete barriers. The increased permeability of Sertoli cell tight junctions after efferent ductule ligation, which caused rapid testicular weight gain followed by atrophy, indicates that tight junctions are sensitive to enforced retention of testicular secretions inside the seminiferous tubules. The apparent normalcy of Sertoli cell tight junctions after vasoligation, which had no effect on testis weight, supports the view that blockage of testicular secretions distal to the epididymis is relatively innocuous.
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