Characterization of granulosa cell subpopulations from avian preovulatory follicles by multiparameter flow cytometry

The objective of the present study was to characterize the granulosa cell populations from individual hen (Gallus domesticus) preovulatory follicles at defined stages of follicular maturation using multiparameter flow cytometry. Granulosa cells were fixed and stained with three fluorochromes that selectively bind to DNA (Hoechst 33342, blue), RNA (pyronin Y, red), or protein (fluorescein isothiocyanate, green). A flow cytometer equipped with a three-laser excitation system was used to analyze three colors of fluorescence from stained cells. Forward angle light scatter and axial light loss measurements were made on each cell to determine relative cell size. In addition, the ratios of RNA to protein and DNA to protein were measured. The major findings obtained from correlated measurements of cell cycle (DNA), protein, RNA, cell size, and ratios were: 1) the percentage of proliferating cells decreased while cell size increased during follicular maturation; 2) two subpopulations of granulosa cells were identified within each follicle based on relatively high and low protein contents; the fraction of cells in the high protein subpopulation increased, and the fraction of cells in the low protein subpopulation decreased during follicular maturation; 3) the high and low protein subpopulations also differed in cell cycle distribution, RNA content, and cell size; and 4) the distribution of cells into the two subpopulations and the degree of proliferation were influenced by stage of the ovulatory cycle, primarily in the most mature follicles. The results demonstrate the dynamic heterogeneity of the granulosa cell populations from individual ovarian follicles and show the influences of follicular maturation and stage of the ovulatory cycle on cell growth and metabolism.     

Evidence for prenatal competition among the central arbors of trigeminal primary afferent neurons.

Previous studies have shown that damage to vibrissa follicles in newborn rats and mice does not alter the brainstem representations of the remaining vibrissa as demonstrated by staining for mitochondrial enzymes such as cytochrome oxidase (CO) succinic dehydrogenase. This study asked whether this lack of effect might be due to the fact that the trigeminal primary afferents in rodents are already quite well developed at birth. We assessed this possibility by using CO staining the evaluate patterns in the brainstems of pre- and postnatal rats. A vibrissa-related pattern began to emerge in trigeminal nucleus principalis and subnucleus interpolaris (Spl) by embryonic day (E-) 19 and appeared fully developed by the day of birth (P-0). We also made partial lesions of the vibrissa pad on E-15-20 and on P-0, killed pups on P-5-7, and measured the size of the CO-stained patches in Spl on both sides of the brainstem. The correspondence between CO patches and clusters of primary afferent terminal arbors was verified in some animals by combining transganglionic horseradish peroxidase tracing and CO staining. Vibrissa pad damage on E-15-18 resulted in significant (20.1-36.9%) increases in the average area of the remaining CO patches in Spl ipsilateral to the lesion. Vibrissa pad damage on E-19, E-20, and P-0 produced small (6.2-8.9%), but insignificant, increases in patch size in Spl ipsilateral to the lesion. We used anatomical and electrophysiological methods to determine whether our lesions altered the trigeminal innervation of surviving vibrissa follicles. We recorded single trigeminal ganglion cells from 12 rats that sustained vibrissa pad lesion on E-17. As in normal rats, all of the 49 vibrissa-sensitive ganglion cells isolated in the lesioned animals were responsive to deflection of one and only one vibrissa. We also dissected 11 deep vibrissal nerves from intact follicles in adult rats that sustained fetal vibrissa pad damage on E-17, and counted numbers of myelinated axons in 1 microns plastic sections. These data were compared with counts from corresponding follicles on the intact side of the face. The average number of myelinated axons innervating follicles in the damaged vibrissa pads was 196.8 +/- 27.9, and that for the corresponding contralateral nerves was 194.6 +/- 25.7. These data suggest that competitive interactions among the central arbors of trigeminal primary afferents in fetal life may influence the development of central vibrissa representations and, further, that lesion-induced central changes need not be correlated with alterations in the peripheral innervation of undamaged follicles.     

The source of the transient serotoninergic input to the developing visual and somatosensory cortices in rat.

For approximately the first two weeks of life, dense serotonin immunoreactivity closely matches the pattern of thalamocortical axons innervating both the granular portion of the primary somatosensory cortex and area 17 in rodents [D'Amato et al. (1987) Proc. natn. Acad. Sci. 84, 4322-4326; Fujimiya et al. (1986) J. comp. Neurol. (1986) 246, 191-201; Rhoades et al. (1990) J. comp. Neurol. 293, 190-207]. This serotonin immunoreactivity is not contained in thalamocortical axons [Rhoades et al. (1990) 293, 190-207] but its source has never been demonstrated. In the present study, a variety of approaches were used to address this issue. The combination of electron microscopy and immunocytochemistry showed that all serotonin immunoreactivity in the developing cerebral cortex was contained in axons and that the terminals of many of these fibers made synapses with the dendrites of cortical cells. Treatment with fluoxetine, a specific inhibitor of serotonin uptake, did not result in a loss of the cortical pattern of serotonin immunoreactivity, indicating that immunoreactive fibers were not labeled solely as a result of serotonin uptake. The combination of retrograde tracing from the primary somatosensory cortex and area 17 with immunocytochemistry demonstrated numerous double-labeled cells in nucleus raphe dorsalis and the median raphe nucleus. Smaller numbers of double-labeled neurons were located in the B9 cell group and the region of the lateral midbrain tegmentum. Large electrolytic lesions that included most of the nucleus raphe dorsalis and median raphe nucleus, but which left the B9 group and more caudal serotoninergic cells undamaged, caused either a substantial reduction in density or complete disappearance of the serotonin pattern in both hemispheres. Unilateral electrolytic lesions of the medial forebrain bundle resulted in a loss of the pattern only on the side of the damage. Injection of the neurotoxin 5,7-dihydroxytryptamine directly into the mesencephalon either abolished or substantially reduced the density of the cortical serotonin immunoreactivity. Injections that produced substantial cell loss in the median raphe nucleus, but only minor cell loss in the nucleus raphe dorsalis had little effect upon the cortical pattern of serotonin immunoreactivity. These results indicate that the dense serotonin immunoreactivity which appears transiently in the visual and somatosensory cortices of perinatal rodents is contained in serotoninergic axons that arise from cells in the nucleus raphe dorsalis and perhaps also the median raphe nucleus.     

Multiparametric deoxyribonucleic acid and cell cycle analysis of breast carcinomas by flow cytometry. Clinicopathologic correlations.

Using intact ethanol-fixed cytokeratin monoclonal (CAM 5.2) and propidium iodide dual-stained cells, we have performed two-color multiparametric flow cytometric (FCM) DNA analysis and S-phase fraction (SPF) determination on 165 mechanically dissociated breast carcinomas. Sixty-seven patients were axillary node positive, 33 patients node negative; 59 had biopsy only and in 8, FCM was performed on tissue from metastatic lesions. Overall, 62% of the tumors contained aneuploid cell populations. Abnormal cellular DNA content (aneuploidy) was significantly correlated with high nuclear grade (p less than 0.001), lack of estrogen receptors (p less than 0.001), presence of vascular invasion (p less than 0.04), high histologic grade (p less than 0.04), and tumor size (p less than 0.03) but not with patient age (p greater than 0.07) or axillary node status (p greater than 0.50). SPF values derived from ungated histograms had a positively skewed frequency distribution (range 2 to 30%, N = 152) with an overall median of 11% (diploid, 8.9%; aneuploid, 15.7%). Higher SPF values were significantly correlated with aneuploidy (p less than 0.001), presence of necrosis (p less than 0.001), lack of estrogen receptor (p less than 0.0001), high nuclear grade (p less than 0.001), vascular invasion (p less than 0.003), tumor size (p less than 0.006), and high histologic grade (p less than .004) but not the presence of lymph node metastases (p greater than 0.56). Mean SPF values were significantly higher when calculated from cytokeratin gated DNA histograms (14.1% versus 11.5%, p less than 0.001), probably due to exclusion of contaminating stromal/inflammatory cells; and significantly lower when calculated from debris subtracted histograms (7.8% versus 11.4%). Cytokeratin gated and debris subtracted SPF values both had a greater degree of correlation than ungated values with clinicopathologic factors of known prognostic significance.(ABSTRACT TRUNCATED AT 250 WORDS)     

Synaptic organization of damaged infraorbital nerve axons in perinatal rats: demonstration by galanin immunocytochemistry

Neonatal transection of the infraorbital nerve (ION; the trigeminal, V, branch that supplies the mystacial vibrissae follicles) results in an upregulation of galanin in the central arbors of primary afferent axons. The present study was undertaken to evaluate the synaptic organization of these galanin-positive primary afferents and compare it with that of normal neurobiotin/biocytin-labeled primary afferent axons from animals of the same age. Examination of 1200 neurobiotin/biocytin-labeled profiles in V nucleus principalis (PrV) of rats killed on postnatal day (P-) 7 indicated that 23.3% (n=279) of these profiles made synaptic contacts: 87.4% were axodendritic, 8.9% were axoaxonic, 2.8% were axosomatic, and 0.7% were axospinous. Evaluation of 1200 galanin-positive profiles in PrV from rats that sustained transection of the ION on P-0 and were killed on P-7 indicated that only 64 (5.3%) of these profiles made synaptic contacts (P<0.05 compared with the intact animals). Of the galanin-positive profiles that did make synapses in PrV, 81.2% (n=52) were axodendritic and 18.8% (n=12) were axoaxonic. These results indicate that galanin released by damaged ION primary afferents in PrV is likely to affect the activity of second-order V neurons by a paracrine action rather than by acting at specific synapses.     

Screening methods for thyroid hormone disruptors

The U.S. Congress has passed legislation requiring the EPA to implement screening tests for identifying endocrine-disrupting chemicals. A series of workshops was sponsored by the EPA, the Chemical Manufacturers Association, and the World Wildlife Fund; one workshop focused on screens for chemicals that alter thyroid hormone function and homeostasis. Participants at this meeting identified and examined methods to detect alterations in thyroid hormone synthesis, transport, and catabolism. In addition, some methods to detect chemicals that bind to the thyroid hormone receptors acting as either agonists or antagonists were also identified. Screening methods used in mammals as well as other vertebrate classes were examined. There was a general consensus that all known chemicals which interfere with thyroid hormone function and homeostasis act by either inhibiting synthesis, altering serum transport proteins, or by increasing catabolism of thyroid hormones. There are no direct data to support the assertion that certain environmental chemicals bind and activate the thyroid hormone receptors; further research is indicated. In light of this, screening methods should reflect known mechanisms of action. Most methods examined, albeit useful for mechanistic studies, were thought to be too specific and therefore would not be applicable for broad-based screening. Determination of serum thyroid hormone concentrations following chemical exposure in rodents was thought to be a reasonable initial screen. Concurrent histologic evaluation of the thyroid would strengthen this screen. Similar methods in teleosts may be useful as screens, but would require indicators of tissue production of thyroid hormones. The use of tadpole metamorphosis as a screen may also be useful; however, this method requires validation and standardization prior to use as a broad-based screen.     

Prostate cancer old problems and new approaches

Rates of prostate cancer (PCa) have increased so dramatically over the last decade that the age adjusted incidence rate for PCa is now greater than that any other cancer among men in the United States. This review, published as a three part series, provides a state-of-art assessment of the PCa problem in its divergent aspects.Part 1 covers epidemiology, incidence and progression. Several epidemiological studies have demostrated that first degree male relatives of men with PCa are at increased risk of developing the disease. Familial and genetic factors as well as medical, anthropometric, dietary, hormonal and occupational factors involved in PCa are discussed. Postmortem examination of the prostate in men without evidence of PCa documented a high frequency of adenocarcinoma. Latent disease occurred as early as the second decade of life. Although there is no significant difference in incidence between Caucasian and African-American males, high grade prostatic intraepithelial neoplasia (HGPIN) is higher in the latter group. While dietary fat, androgens and certain environmental factors may be determinants for PCa, the exact mechanism of tumorigenesis is still relatively unknown. The current thinking of the role of genomic instability, chromosomal alterations, tumor suppressor genes and the androgen receptor are explored.     

Prognostic significance of DNA ploidy in Ta/Tl bladder cancer: A southwest oncology group study

While 80% of transitional cell carcinomas (TCC) present as Ta Tl lesions, they account for only 15% of deaths caused by TCC. We have evaluated the ability of DNA ploidy analysis to predict outcome in 228 patients with Ta Tl TCC. All patients were judged to be at increased risk for tumor recurrence due to having two occurrences of Stage TI tumor within 56 weeks, or three or more tumors presenting simultaneously within 16 weeks of registration. Concurrent carcinoma in situ was acceptable. All patients were treated with either bacillus Calmette Guerin (BCG) immunotherapy or mitomycin-C (MMC) intravesical chemotherapy. Patients with nondiploid tumors had higher hazard rates for both tumor progression and death (p = 0.007 and p = 0.016, respectively); however, the prognostic information of DNA ploidy was not additive to tumor grade.