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.