Distribution of extrajunctional acetylcholine receptors on a vertebrate muscle: evaluated by using a scanning electron microscope autoradiographic procedure

A scanning electron microscope (SEM) autoradiographic technique was calibrated and used to determine the site density of acetylcholine receptors within 250 micron of the neuromuscular junction in innervated as well as 3- and 10-d denervated sternomastoid muscle of the mouse. In all these groups sharp gradients of receptor site density are seen around the endplates in the first 2-7 micron, continuing less sharply to between 25 and 50 micron. Beyond 50 micron (to 250 micron) a spatial density gradient is present 3 d after denervation, but none exist by 10 d. These results suggest that the postdenervation steady-state extrajunctional receptor site density is reached sooner near the junction than away from the junction. The usefulness of SEM autoradiography to study the expression and distribution of membrane molecules at high resolution is demonstrated.     

Rate of DNA replication and size of replicons in human diploid cells]

The rate of DNA replication and the distances between initiation sites (size of replicons) have been studied in human cultured fibroblasts. The modified Huberman and Riggs technique of DNA fiber autoradiography has been used: the pulse-labelled regions were analysed in DNA fibers preliminarily labelled along the whole length. This enabled us: a) to analyse the arrangement of replicons along the length of labelled DNA fibers with the lengths of 200-750 micron, reaching 2700 micron in some cases; b) to select only single DNA molecules for the analysis. This technique decreases the danger of a mistake when minor labelled regions belonging to different DNA molecules are referred to the same one. The rate of DNA replication varies from 0.2 to 1.2 micron/min, the average of 0.6 micron/min. This conforms with findings of other authors. The distances between initiation sites vary from 15 to 140 micron with the modal interval of 50-60 micron. This value is twice higher than those obtained by other authors. The possible reasons for such difference are discussed.     

Platelet-derived growth factor: morphologic and biochemical studies of binding, internalization, and degradation

Kinetic studies of binding and internalization of 125I-platelet-derived growth factor (PDGF) demonstrate that up to 15% of membrane-associated radioactivity is internalized within 2 minutes after warming to 37 degrees C in a variety of cell types. The T 1/2 for internalization is approximately 20 minutes. The T 1/2 for the subsequent appearance of degradation products in the culture medium is between 60-90 minutes following initiation of internalization. Internalization and lysosomal association of 125I-PDGF were confirmed by EM autoradiography. Quantitative studies using PDGF adsorbed to colloidal gold (gold-PDGF) demonstrate that 17% of the cell-associated sites are along coated regions of the plasma membrane (1.0 sites/micron), while 82% are associated with noncoated membrane (0.2 sites/micron). There is a significant redistribution of the gold-PDGF complexes upon warming. Within 1-2 minutes at 37 degrees C, gold particles are found within endocytic vesicles, endosomes (0.09-0.3 micron diameter), and lysosomes (greater than 0.2 micron diameter). At this time the vesicle/endosome compartment comprises 15% of the total sites and contains 0.9 sites per micron2 of surface area. The lysosomes account for 8% of the total sites and contain 0.8 sites per micron2 of surface area. Simultaneously, there is an increase in the number of gold-PDGF binding sites within coated-pits (1.6 sites/micron, 18% of the total sites) and a decrease along noncoated regions of the membrane (0.11 sites/micron, 58% of the total sites). After 15 minutes at 37 degrees C, 26% of the total sites (1.4 sites/micron2) are highly concentrated within lysosomes, while sites in the vesicle/endosome compartment remain constant. At the same time, binding sites within coated pits decrease substantially (0.5 sites/micron, 4% of the total sites), while the number of sites along noncoated regions of the membrane remain constant. Gold-PDGF was not observed associated with the Golgi complex at any time up to 120 minutes following warming. We conclude that gold-PDGF is processed via both receptor-mediated and nonspecific endocytosis and follows an intracellular pathway comparable to that followed by some other protein ligands.     

Mechanism of thymidylate synthase inhibition by methotrexate in human neoplastic cell lines and normal human myeloid progenitor cells

We have studied the roles of 5,10-methylenetetrahydrofolate (5,10-methylene-H4PteGlu) depletion and dihydrofolate (H2PteGlu) accumulation in the inhibition of de novo thymidylate synthesis by methotrexate (MTX) in human MCF-7 breast cancer cells. Using both a high pressure liquid chromatography system and a modification of the 5-fluoro-2'-deoxyuridine-5'-monophosphate radioenzymatic binding assay, we determined that the 5,10-methylene-H4PteGlu pool is 50-60% depleted in human MCF-7 breast cancer cells following exposure to 1 micron MTX for up to 21 h. Similar alterations in the 5,10-methylene-H4PteGlu pools were obtained when human promyelocytic HL-60 leukemia cells and normal human myeloid precursor cells were incubated with 1 micron MTX. The H2PteGlu pools within the MCF-7 cells increased significantly after 15 min of 1 micron MTX exposure, reaching maximal levels by 60 min. Thymidylate synthesis, as measured by labeled deoxyuridine incorporation into DNA, decreased to less than 20% of control activity within 30 min of 1 micron MTX exposure. The inhibition of thymidylate synthesis coincided temporally with the rapid intracellular accumulation of H2PteGlu, a known inhibitor of thymidylate synthase. Furthermore, inhibition of this pathway was associated in a log-linear fashion with the intracellular level of dihydrofolate. These studies provide further evidence that depletion of the thymidylate synthase substrate 5,10-methylene-H4PteGlu is inadequate to account completely for diminished thymidylate synthesis resulting from MTX treatment. Our findings suggest that acute inhibition of de novo thymidylate synthesis is a multifactorial process consisting of partial substrate depletion and direct enzymatic inhibition by H2PteGlu polyglutamates.     

Comparison of fluorographic methods for detecting radioactivity in polyacrylamide gels or on nitrocellulose filters

The commercial fluorographic enhancers, En3Hance or Amplify, were not as efficient as 2,5-diphenyloxazole (PPO) for detecting radioactively labeled proteins in polyacrylamide gels or on nitrocellulose filters. For most of the X-ray films tested, optimal preexposure was essential to obtain maximum sensitivity in fluorography or indirect autoradiography using intensifying screens. The best results were obtained with nitrocellulose by saturating the filters with PPO. The minimum levels of 35S/14C that could be detected on filters by autoradiography or fluorography in a 24-h exposure were 4 X 10(2) or 1 X 10(2) dpm cm-2 respectively. For 3H these levels were, respectively, 20 X 10(3) or 0.5 X 10(3) dpm cm-2.     

Chromosomal effects of carcinogens and non-carcinogens on WI-38 after short term exposures with and without metabolic activation.

The human diploid fibroblast culture, WI-38 was analyzed for chromosomal damage after 24 h exposures to benzo(a)pyrene (BP), 3-methylcholanthrene (MCA), n-methyl-n'-nitrosoguanidine (MNNG), 4-nitroquinoline-1-oxide (4NQO), pyrene and caffeine. A low concentration of 4NQO (0.15 micron) and MNNG (1.9 micron) produced breakage and exchange figures. A relatively high concentration of caffeine (1300 micron) caused breakage. The other compounds (BP, MCA and pyrene) caused little or no increase in damage above the control levels. A 1-h pulse exposure of WI-38 cells to BP (40 micron) in the presence of a rat liver homogenate supernate (S-9) resulted in damage significantly greater than the untreated cells or cells treated with BP alone. 4NQO (0.25 micron) produced exchange figures after a similar 1-h exposure, but this effect was eliminated by the S-9. A much higher concentration of caffeine (10,300 micron) was required to cause breakage greater than control levels after a one hour exposure. The results indicate a possible short term in vitro human cell system for distinguishing carcinogens, procarcinogens, and noncarcinogens.     

Light microscopic autoradiographic localization of [3H]pirenzepine and [3H](-)quinuclidinyl benzilate binding in human stellate ganglia

We have utilized the LKB Ultrofilm method of autoradiography to anatomically localize putative M1 and M2 muscarinic receptor subtypes in human stellate ganglia. Ten micron sections were labeled in vitro with either 1 nM of the classical antagonist [3H](-)quinuclidinyl benzilate ([3H](-)QNB) or 20 nM of the non-classical antagonist [3H]pirenzepine ([3H]PZ), using 1 microM atropine sulfate to define non-specific binding for both ligands. Our results indicate that [3H](-)QNB and [3H]PZ binding sites are distributed within the principal ganglion cells and nerve bundles.     

Regulation of cyclic nucleotide phosphodiesterase activity in human lung fibroblasts.

Cyclic nucleotide phosphodiesterase activity (3', 5'-cyclic-nucleotide 5'-nucleotidohydrolase, 3.1.2.17) was studied in homogenates of WI-38 human lung fibroblasts using 0.1--200 microgram cyclic nucleotides. Activities were observed with low Km for cyclic AMP(2--5 micron) and low Km for cyclic GMP (1--2 micron) as well as with high Km values for cyclic AMP (100--125 micron) and cyclic GMP (75--100 micron). An increased low Km cyclic AMP phosphodiesterase activity was found upon exposure of intact fibroblasts to 3-isobutyl-1-methylxanthine, an inhibitor of phosphodiesterase activity in broken cell preparations, as well as to other agents which elevate cyclic AMP levels in these cells. The enhanced activity following exposure to 3-isobutyl-1-methylxanthine was selective for the low Km cyclic AMP phosphodiesterase since there was no change in activity of low Km cyclic GMP phosphodiesterase activity or in high Km phosphodiesterase activity with either nucleotide as substrate. The enhanced activity due to 3-isobutyl-1-methylxanthine appeared to involve de novo synthesis of a protein with short half-life (30 min), based on experiments involving cycloheximide and actinomycin D. This activity was also enhanced with increased cell density and by decreasing serum concentration. Studies of some biochemical properties and subcellular distribution of the enzyme indicated that the induced enzyme was similar to the non-induced (basal) low Km cyclic AMP phosphodiesterase.     

In vivo autoradiographic demonstration of beta-adrenergic binding sites in adult rat type II alveolar epithelial cells

Adult male rats were injected intravenously with the muscarinic binding probe 3H-Quinuclidinyl benzilate (QNB) or the beta-adrenergic probe 3H-dihydroalprenolol (DHA). Other rats were pre-treated with an intraperitoneal injection of a 500-fold excess of L-isoproterenol prior to the DHA. Light microscopic autoradiography of 0.5 micron sections of lung from the QNB group demonstrated very little labelling even after 6 months of exposure. In contrast, trachealis smooth muscle from these animals contained substantial labelling. Autoradiographs of lung from rats injected with DHA demonstrated labelling which was well localized over alveolar septa and concentrated over the cytoplasm of type II cells. Quantitative analysis of labelling in the DHA groups indicated a significant reduction of labelling in animals treated with L-isoproterenol prior to DHA, in both the alveolar parenchyma in general and over type II cells. The results of this study provide morphologic evidence for the uptake and specific binding of beta-adrenergic antagonists by the adult lung in vivo, while failing to demonstrate similar binding of a muscarinic probe. In addition, the results demonstrate specific beta-adrenergic receptors on type II cells in vivo and substantiate the view of a direct effect of beta-adrenergic agonists on alveolar type II cells.     

Do C cells of the thyroid gland of the baboon contain estrogen receptors?

The uptake and retention of radiolabelled estradiol was studied in the thyroid gland of the female baboon. Four baboons were injected intravenously with 1 micron/kg body weight of 3H-estradiol. One animal, which served as a control, received an additional injection of 100 micrograms/kg body weight of unlabelled hormone. One hour after the injections, the animals were killed and the thyroid glands removed and processed for either autoradiography or autoradiography in combination with immunocytochemical staining for C cells. Localization of estradiol was observed in the nuclei of interstitial cells, but not in those of the follicular cells. Nuclei of immunostained calcitonin-containing cells in both the walls of the follicles and the interfollicular compartment were not radiolabelled. This study suggests that estrogen does not regulate calcitonin secretion by the C cells of the thyroid via a classical receptor system.     

HIGH-RESOLUTION AUTORADIOGRAPHY : I. Methods

Methods used in obtaining high resolution in autoradiography, with special emphasis on the technique of electron microscopic autoradiography, are described, together with control experiments designed to establish the optimum conditions or procedures. On the basis of these experiments the emulsion selected was Ilford L-4, with a crystal size slightly larger than 0.1 micron. It is applied to the specimen in the form of a gelled film consisting of a monolayer of silver halide crystals. Background, when present, can be eradicated by a simple method. The preparations can be stored, in presence of a drying agent, at room temperature or in a refrigerator. Photographic development is done in Microdol, or in a special fine grain "physical" developer. For examination in the electron microscope the sections are stained with uranyl or lead stains. These methods give a good localization of the label, at the subcellular level, and good reproducibility in relative grain counts.     

Quantitative autoradiographic study of FSH binding sites in prepubertal ovaries of three strains of rats

Iodinated FSH was injected to 18- and 36-day-old rats of 3 strains (03, 04 and 12) with different sensitivity to FSH (12 less than 03 less than 04) and autoradiography was performed on histological sections of the labelled ovaries. Specific labelling was quantified by microphotometry on histological slides, on granulosa cells of individual follicles with different sizes (greater than 80 micron diameter) and qualities. In small preantral follicles (less than 160 micron diameter) the labelling was low and homogeneous within the granulosa; it increased between 18 and 36 days of age in the 3 strains. At 36 days, ovaries were characterized by the presence of large preantral and antral follicles with a higher labelling in the outer layers of granulosa (near the theca), compared to the inner layers. In definitely atretic follicles, a loss of binding sites was detected in the outer layers. In rats of Strains 03 and 04, the number of binding sites for FSH in the outer layers of granulosa of follicles with a diameter of greater than 160 micron increased with follicular size; no change was detected in follicles of Strain 12 rats. The low number of binding sites for FSH and the lack of terminal maturation which characterize the follicles of strain 12 rats can be related to the poor and delayed follicular development, the low sensitivity to exogenous FSH and the low fertility of the animals of this strain.     

Acetylcholinesterase in the fast extraocular muscle of the mouse by light and electron microscope autoradiography

The distribution of acetylcholinesterase (ACHe) in the twitch fibers of the extraocular muscles of the mouse was examined by light and electron microscope autoradiography after labeling with radioactive diisopropyl fluorophosphate (DFP) with, and without, 2-pyridine aldoxime methiodide (2-PAM) reactivation. The values obtained were compared with those previously reported for the diaphragm and sternomastoid muscles. The extraocular muscles were studied because they differ from the other two muscles in that they are among the fastest of the mammalian muscles, yet their endplates have sparse junctional folds. They could thus provide information on the extent to which ACHe concentration is an invariant feature of endplate morphology and what, if any aspects may be related to their fast speed of response. We found, using light microscope autoradiography, that in the twitch fibers of the extraocular muscle, there is n average of 6.4 +/- 2.1 X 10(7) DFP- binding sites per endplate, of which 29% (1.8 X 10(7)) are reactivated by 2-PAM and are thus AChe. The morphology of the extraocular endplates allowed us to conclude, on statistical grounds, that the AChe site are probably localized not only along the surface area of the postjunctional membrane (PJM) but also along the surface of the presynaptic axonal membrane. Based on this localization, we calculate 7,800 DFP sites and 2,500 2-PAM-reactivated sites/micron 2 of surface area of pre-and postjunctional membrane. This stacking density of DFP- binding sites per surface area of membrane ( probably in the overlying sheets of basal lamina) is very similar to that in the diaphragm and sternomastoid muscles.     

Enrichment and visualization of small replication units from cultured mammalian cells

DNA from cultured Chinese hamster cells has been fractionated to yield a population of DNA enriched for replicating molecules. Molecules containing replication structures were analyzed by electron microscopy, and replicon size was estimated. The enrichment procedure takes advantage of single-stranded regions characteristic of replicating molecules, and the greater affinity of mercuric ion for single-stranded rather than native DNA. After interaction with low concentrations of HgCl2, DNA with bound mercury is separated from the bulk of the DNA by virtue of its increased buoyant density in an isopycnic Cs2SO4 gradient. When DNA from cells labeled with [3H]thymidine for 45 s is interacted with HgCl2 and banded in Cs2SO4, the DNA with the highest specific activity is found in a dense region of the gradient. The high specific activity DNA behaves kinetically like nascent DNA since the radioactivity can be chased into main band if the cells are incubated for a further 2 h in excess unlabeled thymidine. Electron microscope analysis of the DNA in the enriched fraction confirmed that it contains a substantial fraction of molecules with replication structures. The level of enrichment is about 25-fold compared to unfractionated DNA or DNA taken from the main band of the Hg++/Cs2SO4 gradient. Of the replicating molecules visualized, 85% possessed a single replication structure. All molecules with multiple replication forms contained replicon sizes less than 5 micron, ranging from 0.2 to 4.5 micron. Replicon size was determined by measuring the distance from the center of one replication structure to the center of the adjacent replication structure on the same molecule. The replicons observed in this study are far smaller than can be detected by DNA fiber autoradiography and are in the same size range as the very small replication units reported in embryonic systems.     

Replication of each copy of the yeast 2 micron DNA plasmid occurs during the S phase.

Saccharomyces cerevisiae contains 50-100 copies per cell of a circular plasmid called 2 micron DNA. Replication of this DNA was studied in two ways. The distribution of replication events among 2 micron DNA molecules was examined by density transfer experiments with asynchronous cultures. The data show that 2 micron DNA replication is similar to chromosomal DNA replication: essentially all 2 micron duplexes were of hybrid density at one cell doubling after the density transfer, with the majority having one fully dense strand and one fully light strand. The results show that replication of 2 micron DNA occurs by a semiconservative mechanism where each of the plasmid molecules replicates once each cell cycle. 2 micron DNA is the only known example of a multiple-copy, extrachromosomal DNA in which every molecule replicates in each cell cycle. Quantitative analysis of the data indicates that 2 micron DNA replication is limited to a fraction of the cell cycle. The period in the cell cycle when 2 micron DNA replicates was examined directly with synchronous cell cultures. Synchronization was accomplished by sequentially arresting cells in G1 phase using the yeast pheromone alpha-factor and incubating at the restrictive temperature for a cell cycle (cdc 7) mutant. Replication was monitored by adding 3H-uracil to cells previously labeled with 14C-uracil, and determining the 3H/14C ratio for purified DNA species. 2 micron DNA replication did not occur during the G1 arrest periods. However, the population of 2 micron DNA doubled during the synchronous S phase at the permissive temperature, with most of the replication occurring in the first third of S phase. Our results indicate that a mechanism exists which insures that the origin of replication of each 2 micron DNA molecule is activated each S phase. As with chromosomal DNA, further activation is prevented until the next cell cycle. We propose that the mechanism which controls the replication initiation of each 2 micron DNA molecule is identical to that which controls the initiation of chromosomal DNA.     

Distribution of label after intrathecal administration of 125I-substance P in the rat

Despite the widespread use of the intrathecal route for the administration of neuroactive agents, little is known about the penetration of these agents into the spinal cord. In the present study, 125I-substance P was injected via a spinal catheter to the thoracic or sacro-coccygeal spinal cord in the rat (350-400 g) anesthetized with urethane (2.5 g/kg). Spinal cords were removed rapidly at 1 or 10 min after injection and immediately frozen in CCl2F2. Frozen sections, 20 micron thick, were cut and mounted for autoradiography. Autoradiographs of transverse sections demonstrated that the label penetrated 700 to 1800 micron from the surface of the spinal cord at both levels. In longitudinal sections, this penetration extended about 0.5 cm rostrally and caudally from the site of injection. Serial autoradiographs of transverse sections showed a similar penetration rostro-caudally. In addition, venous blood samples were taken at 1, 6, 11 and 16 min after injection of the labelled peptide. Quantification of the radioactivity in the samples revealed that 0.8 to 3.5% of the total CPM injected had passed into the general circulation at these times. These data indicate that after intrathecal administration of radiolabelled substance P, the label penetrates into the grey matter of the spinal cord to presumed sites of action. They also suggest that the rostro-caudal extent of penetration is more localized than suggested from earlier studies which looked only at levels of radioactivity in pieces of whole spinal cord. Finally, our study has indicated that passage of label into the circulation is negligible at least for substance P.(ABSTRACT TRUNCATED AT 250 WORDS)     

Androgen receptor localization in the human prostate: demonstration of heterogeneity using a new method of steroid receptor autoradiography

We have used a novel receptor labeling and autoradiographic technique to identify the cell types in human benign prostatic hyperplasia (BPH) that contain androgen receptors, and we have found that androgen receptor localization is heterogeneous. Prostatic androgen receptors were labeled by incubating slide-mounted frozen tissue sections (10 micron thickness) with [3H]R1881 in vitro. Tissue sections labeled in this way were subjected to concurrent biochemical and autoradiographic analysis. Some of the sections were wiped from the slides for scintillation counting to validate that the procedure indeed measures total cellular androgen receptors of appropriate high affinity and androgen steroid specificity. Replicate labeled slide-mounted tissue sections were dried rapidly, apposed to dry emulsion-coated coverslips, and exposed in the dark for autoradiography. Autoradiograms were developed, fixed, and stained; silver grains were counted over nuclei or cytoplasm of epithelium or stroma to evaluate specific androgen receptor location. Autoradiographic analysis of human glandular BPH demonstrated androgen receptor localization almost exclusively in the epithelial nuclei, with little or none in the stroma. We anticipate that data obtained using this new method of steroid receptor autoradiography may provide fresh insight into the mechanism of hormonal regulation of the prostate.     

Characterization of small and large human peripheral blood monocytes: effects of in vitro maturation on hydrogen peroxide release and on the response to macrophage activators

Human peripheral blood monocytes (HPBM) isolated from normal donors by centrifugal elutriation were divided into two populations according to volume. (Median volumes of small monocytes (SM) and large monocytes (LM) were 255 micron and 280 micron, respectively.) H2O2 production was determined during in vitro culture and in response to bacterial lipopolysaccharide (LPS), and to recombinant human interferon-gamma (rIFN-gamma). On day 1, H2O2 production by LM was significantly greater than that by SM. In vitro culture of SM resulted in an augmented ability to produce H2O2. By day 3, SM were the major H2O2 producers. Freshly isolated SM and LM, exposed for 24 hr to LPS and rIFN-gamma, showed different patterns of activation. Both SM and LM responded to LPS, with LM responding maximally at lower doses than SM. Only SM showed a significant augmentation of H2O2 production with rIFN-gamma treatment. We also assessed the effect of in vitro culture with activation. SM but not LM showed an increased H2O2 to LPS and rIFN-gamma after 7 days in culture. Continuous exposure of SM to rIFN-gamma resulted in maximal H2O2 production at day 3 of culture; this pattern was not seen for LPS. The production of H2O2 by HPBM is related to in vitro maturation. The enhanced H2O2 production by HPBM upon exposure to rIFN-gamma may be related to the induction of in vitro maturation.     

DNA replication fork progression rate and temporal organization of S phase in normal epidermis and in basal cell carcinoma

The double-pulse labeling technique for DNA fiber autoradiography was applied to epidermal cells from normal human skin and from human basal cell carcinoma (BCC). We aimed to measure the size and replication rate of the replication unit (RU) for both types of cell and to account, from these results, for our previous observation of a near doubling of S-phase duration in BCC, compared with normal skin. The mean RU size was 76 +/- 4 micron in BCC, not significantly different from the 68 +/- 6 micron value found in normal skin, so the mean of those two values (i.e., 72 micron), was used in further calculations. The rate of replication fork progression was 0.59 +/- 0.005 micron/min in the normal epidermis and 0.33 +/- 0.03 micron/min in BCC, corresponding to a replication time of the average RU equal to 61 min and 109 min, respectively. Thus, with an unchanged RU size in BCC, the observed 1.8-fold decrease in the rate of fork progression in the tumor can account entirely for our previous observation of a 1.8-fold increase in S-phase duration in this tumor, without requiring the assumption of any change in the temporal organization of DNA synthesis in the malignant cells. Considering S phase as an ordered process in which a major part, if not all, of the genome replicates at genetically determined times, we suggest that the clusters of replication units are, in turn, organized into temporally defined "sets". These sets are composed of all the clusters (whatever their chromosomal location) that are programmed to initiate replication during the same fraction of the S period. This hypothesis implies that DNA synthesis in a given set is triggered by some event coupled to progression of replication in the immediately preceding set. Based on a S-phase duration of 10.2 hours in normal skin and of 19.2 hours in BCC (our previous data), and assuming perfect synchrony and homogeneity of the clusters within each set and of each cluster's constitutive RUs, the minimum number of sequentially replicating sets, in both instances, can be estimated as roughly equal to 10.     

Delayed maturation of the male cerebral cortex in rats.

45 male and female Wistar rats were given a single injection of 3H-thymidine (10 mu Ci/g body weight) on day 1, 7, 14 or 21. All animals survived until 60 days of age when they were perfused with 10% neutral formalin and the brains were removed and prepared for autoradiography. The sagittal section of the cortex (L980 micron) was 6.8% larger in the males (p less than 0.05) but the packing density of the cortical cells was 5.9% higher in the females (p less than 0.01), thus bringing the total number of cells to the male levels. The diameter of the female cortical cells was 3.8% smaller than those of the males (p less than 0.05). The greatest difference was among the smaller cells (3-9 micron). The rate of postnatal acquisition of cortical cells was indicated by the number of radioactive-labelled cells. Males had more labeled cells after each injection; it was most pronounced (32% difference) on day 7 (p less than 0.05). This may reflect a delayed acquisition rate of cells formed before birth, since more cells could be labeled by the postnatal injection.