Postnatal development of the dentate gyrus: a karyometric and topographic study.

We have analysed the postnatal development of the nuclear sizes of the granular cells of the dentate gyrus in 5- to 190-day-old male mice. The study was performed in three topographic levels: rostral, intermediate and caudal. Three subdivisions were analysed in each level: suprapyramidal blade, infrapyramidal blade and the transition between them, the angular zone. Additionally, each of these subdivisions was measured in its external and internal layer, separately. Three gradients of postnatal karyometric development can be described: external-to-internal, suprapyramidal-to-infrapyramidal, and caudal-to-rostral, indicating that the external, suprapyramidal and caudal cells show higher karyometric sizes than the other subdivisions. These gradients are related to the ontogenetic gradients of these neurons.     

Development of the subfornical organ and area postrema of the male albino mouse. Karyometric effect of neonatal and prepuberal castration

We have studied the karyometric development of the nuclei of the ependymal cells and neurons of the subfornical organ and the area postrema in the male albino mouse from the 5th to the 190th postnatal day. We have found similar patterns of development in both although the area postrema showed more significant postnatal oscillations than those of the subfornical organ, suggesting a more intimate chronological relationship to gonadal development. We have furthermore analyzed the development in two experimental groups: in the one animals were castrated at birth, in the other, castration was made on the 20th postnatal day. We have found that neonatal castration produced a significant decrease of nuclear sizes; this was more evident in the subfornical organ than in the area postrema in earlier stages of development while the response was similar in both at peripuberal ages. The response to prepuberal castration was similar in both organs.     

The timing of hamster sperm nuclear decondensation and male pronucleus formation is related to sperm nuclear disulfide bond content

The relationship between the timing of both sperm nuclear decondensation and male pronucleus formation in the oocyte and the relative level of disulfide bonds within the sperm nucleus was evaluated. Since reduction of sperm nuclear disulfide (S-S) bonds is a prerequisite for sperm nuclear decondensation in vitro and in vivo, we hypothesized that sperm nuclei with relatively few S-S bonds would require less time to decondense in the oocyte than sperm nuclei with higher numbers of S-S bonds, and that male pronucleus formation would occur more rapidly as well. Four types of hamster sperm nuclei, in which the extent of S-S bonding differed, were microinjected into hamster oocytes, and the time course of sperm nuclear decondensation and male pronucleus formation was charted. Cauda epididymal sperm nuclei, which are rich in S-S bonds, required 45-60 min to decondense. In contrast, nuclei containing few S-S bonds (namely sonication-resistant spermatid nuclei and cauda epididymal sperm nuclei treated in vitro with the S-S bond-reducing agent dithiothreitol) decondensed within 5-10 min of microinjection. Caput epididymal sperm nuclei, with intermediate S-S bond content, decondensed in 10-20 min. Regardless of when decondensation occurred, formation of the male pronucleus never preceded that of the female pronucleus, which occurred 1.25-1.5 h after microinjection. However, sperm nuclei with few S-S bonds were more likely than S-S rich nuclei to transform into male pronuclei in synchrony with the formation of the female pronucleus. We conclude that the timing sperm nuclear decondensation and pronucleus formation depends in part upon the S-S bond content of the sperm nucleus.     

Reversible chromosome condensation induced in Drosophila embryos by anoxia: visualization of interphase nuclear organization

We have studied the morphology of nuclei in Drosophila embryos during the syncytial blastoderm stages. Nuclei in living embryos were viewed with differential interference-contrast optics; in addition, both isolated nuclei and fixed preparations of whole embryos were examined after staining with a DNA-specific fluorescent dye. We find that: (a) The nuclear volumes increase dramatically during interphase and then decrease during prophase of each nuclear cycle, with the magnitude of the nuclear volume increase being greatest for those cycles with the shortest interphase. (b) Oxygen deprivation of embryos produces a rapid developmental arrest that is reversible upon reaeration. During this arrest, interphase chromosomes condense against the nuclear envelope and the nuclear volumes increase dramatically. In these nuclei, individual chromosomes are clearly visible, and each condensed chromosome can be seen to adhere along its entire length to the inner surface of the swollen nuclear envelope, leaving the lumen of the nucleus devoid of DNA. (c) In each interphase nucleus the chromosomes are oriented in the "telophase configuration," with all centromeres and all telomeres at opposite poles of the nucleus; all nuclei at the embryo periphery (with the exception of the pole cell nuclei) are oriented with their centromeric poles pointing to the embryo exterior.     

Uterine estrogen receptor in vivo: phosphorylation of nuclear specific forms on serine residues

We have characterized further the heterogeneous nuclear-specific doublet forms of the mouse uterine estrogen receptor (ER). Estrogen treatment produced the multiple nuclear ER forms of 65 and 66.5 kDa, which were isolated and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Soluble ER preparations exhibited only a single 65-kDa form. Isolation of the individual nuclear ER forms and reanalysis demonstrated that formation of the multiple bands was not due to artifacts of nuclear sample preparation or the presence of contaminating proteins. Analysis of individual uterine cell types (epithelial and stromal/myometrium) indicated that both ER forms were present in both cell fractions. Fractionation of nuclear components with low salt showed that both ER forms were found in the salt-resistant fraction. Extraction of nuclei with high salt (0.6 M KCl) solubilized both ER forms. Phosphorylation was studied as a protein modification to account for the multiple forms. Incorporation of 32P into uterine protein both in vivo and in intact tissue incubation indicated 32P labeling of uterine nuclear ER after hormone treatment. Both nuclear ER forms are labeled, although the 66.5-kDa form appears to be more heavily labeled. Phosphoamino acid analysis of the immunopurified 32P-labeled ER from intact uterine tissue indicated phosphoserine as the only phospholabeled residue. These data suggest that phosphorylation is associated with the physiological functioning of the ER in response to hormone and produces the heterogeneous ER forms in the nucleus.     

Autoradiographic localization of 3Hdihyrotestosterone in the preoptic area,hypothalamus, and amygdala of a male rhesus monkey

In a preliminary study, autoradiography was used to localize target cells for ³Hdihydrotestosterone (DHT), a non-aromatizable androgen, in the brain of the rhesus monkey. One castrated male was injected intravenously with 2 mCi of ³HDHT (0.42 μg/kg), and was killed one hour later. Neurons that concentrated radioactivity in their nuclei were located in widespread areas of the brain, which included the medial and suprachiasmatic preoptic nuclei, bed nucleus of the stria terminalis, lateral septal nucleus, anterior hypothalamic area, ventromedial, arcuate, or dorsemedial, and paraventricular hypothalamic nuclei, ventral premammillary nucleus, and medial, cortical, basal accessory, and lateral amygdaloid nuclei. These results indicate that the topographic distribution of androgen target neurons is considerably wider than that observed in a study using ³Htestosterone (T) in the male rhesus monkey (1). However, further work is needed to elucidate these differences before attempting correlations between behavioral activity and androgen receptors in the brain.     

Remodeling the sperm nucleus into a male pronucleus at fertilization

After fertilization, the dormant sperm nucleus undergoes morphological and biochemical transformations leading to the development of a functional nucleus, the male pronucleus. We have investigated the formation of the male pronucleus in a cell-free system consisting of permeabilized sea urchin sperm nuclei incubated in fertilized sea urchin egg extract containing membrane vesicles. The first sperm nuclear alteration in vitro is the disassembly of the sperm nuclear lamina as a result of lamin phosphorylation mediated by egg protein kinase C. The conical sperm nucleus decondenses into a spherical pronucleus in an ATP-dependent manner. The new nuclear envelope (NE) forms by ATP-dependent binding of vesicles to chromatin and GTP-dependent fusion of vesicles to each other. Three cytoplasmic membrane vesicle fractions with distinct biochemical, chromatin-binding and fusion properties, are required for pronuclear envelope assembly. Binding of each fraction to chromatin requires two detergent-resistant lipophilic structures at each pole of the sperm nucleus, which are incorporated into the NE by membrane fusion. Targeting of the bulk of NE vesicles to chromatin is mediated by a lamin B receptor (LBR)-like integral membrane protein. The last step of male pronuclear formation involves nuclear swelling. Nuclear swelling is associated with import of soluble lamin B into the nucleus and growth of the nuclear envelope by fusion of additional vesicles. In the nucleus, lamin B associates with LBR, which apparently tethers the NE to the lamina. Thus male pronuclear envelope assembly in vitro involves a highly ordered series of events. These events are similar to those characterizing the remodeling of somatic and embryonic nuclei transplanted into oocytes. The relationship between sperm nuclear remodeling at fertilization and nuclear remodeling after nuclear transplantation is discussed.     

The Development of Gynoecium after Anthesis and Fertilization in Eleutherococcus senticosus (Araliaceae)

The development status of gynoecia in Eleutherococcus senticosus flowers is different from that in ordinary plants. Female gametophytes of E. senticosus have not become mature until the 6th day after anthesis. On the 6th day, 82.25% of embryo sacs in female plants, and 67.25％ of those in hermaphroditic plants become mature, while the rest are sterile, immature or degenerated with no fertilized embryo sacs observed. At the same time, all embryo sacs degenerated and flowers withered in male plants. On the 7th day, a few embryo sacs in female and hermaphroditic plants start being fertilized. Accompanying the differentiation of embryo sacs, styles of female and hermaphroditic flowers start to expand and their nectaries become mature gradually. After the 4th or 6th day of anthesis, stigmatic papillae become conspicious and stigmata become white and open. In the meantime, the stigmata become receptive and the nectaries get active or reactive. By the 9th or 10th day, 40～65 ％ of embryo sacs in female plants and 25～41% of those in hermaphroditic plants have been fertilized. The whole process of fertilization in E. senticosus was observed. About 2 or 3 days after pollination, the two sperm nuclei start to fuse with the egg and the secondary nucleus. The fertilization of E. senticosus belongs to the premitotic type of syngamy. The essential process of the fusion of male and female nuclei during syngamy may be generalized as follows: (1) the contacting of male nucleus with the female one; (2) the fusion of nuclear membranes between the male and female nuclei; (3) the despiralization of male spireme and the appearance of male nucleolus inside the fertilized female nucleus; (4) the dispersion of male chromatin and the mergence with the female chromatin, which is the sign of completion of the fusion of the two nuclei. In addition, degeneration types of mature embryo sacs were observed. And typical polyspermy and a series of cases in which extra sperms enter the em-bryo sac are recorded.     

Testosterone 5 alpha-reductase in discrete hypothalamic nuclear areas in the rat: effect of castration

The conversion of testosterone into 5 alpha-dihydrotestosterone (DHT) has been studied in different hypothalamic nuclear areas and in the superficial layers of the cerebral cortex of normal and castrated male rats. The tissue fragments utilized in each incubation have been punched from frozen brain sections utilizing calibrated needles. Castration has been performed 12 (short term) and 180 (long term) days before sacrifice. The nuclear areas studied include: the medial preoptic nucleus (MPN), the lateral preoptic nucleus (LPN), the anterior hypothalamic nucleus (AHN), the lateral hypothalamic nucleus (LHN), the posterior hypothalamic nucleus (PHN), the nucleus ventromedialis (HVM), the arcuate nucleus (AR), the median eminence (ME), the nucleus paraventricularis (HPV), the supraoptic nucleus (SO) and the suprachiasmatic nucleus (SC). The possible effect of castration on the 5 alpha-reductase, were assessed in the MPN,LPN,AHN,LHN,PHN and in the cerebral cortex. The results indicate that, in the male rat: 1) the lateral preoptic(LPN) and the lateral hypothalamic nuclei(LHN) possess a 5 alpha-reductase activity higher than that present in the cerebral cortex and in the other hypothalamic nuclei considered; 2)the suprachiasmatic nucleus (SC) apparently possesses a testosterone metabolizing activity lower than that found in any other nervous structures studied so far; 3) castration does not seem to influence the 5 alpha-reductase activity either in the hypothalamic nuclear structures considered or in the cerebral cortex.     

Influence of the feeding regimen on the rhythmicity of the processes of secretion of the supraoptic nucleus in C57Bl mice

During the period of vernal equinox in Leningrad 2 groups of C57Bl male mice have been investigated. Ninety-five animals are given food ecologically adequate at 9 p.m. Eighty-four animals are given foot at 9 a.m.--ecologically inverted regimen of feeding (IRF). The mice are decapitated for 4 days with an interval about 1.5 h. Serial paraffin sections are stained with aldehyde-fuchsin after Gomori and an additional staining of the nuclei with azocarmine. Criteria for the neurosecretory activity is the ratio of the cells amount at various stages of synthesis, outflux and accumulation of the secrete, volumes of the nuclei and nucleoli. Spectrum and parameters of the rhythmicity are revealed. IRF produces decrease in the amount of the ultradian component of the rhythmic parameters, characterizing active synthesis and discharge of the secrete. The part of the neurosecretory cells, those actively synthesizing and discharging the secrete, and volume of the cell nucleoli decrease. Range of ultradian component of the cell part rhythm, depositing the secrete, and the cell volume enriches. Thus, IRF produces certain changes in the rhythmicity of the cell secretion at all the stages: synthesis, discharge and accumulation of the secrete. Total intensity of the synthetic processes decreases. A conclusion is made that IRF inhibits the microsecretory process in the supraoptic nucleus (SON) and decreases adaptive possibilities of its cells. Adaptation to IRF is performed at the expense of rhythmic discharge of neurohormones, deposited in the cells, and at the expense of processes, occurring in the neuryoplasm and resulting in increase of the nuclear volume.(ABSTRACT TRUNCATED AT 250 WORDS)     

The Corticocuneate Pathway in the Cat: Relations among Terminal Distribution Patterns,Cytoarchitecture, and Single Neuron Functional Properties

A combined anatomical and physiological strategy was used to investigate the organization of the corticocuneate pathway in the cat. The distribution of the corticocuneate projection was mapped by means of the anterograde horseradish peroxidase (HRP) labeling technique and correlated with the nuclear cytoarchitecture in Nissl and Golgi material, the distribution of retrogradely labeled relay cells after HRP injections in the ventrobasal complex of the thalamus, and the topographic organization derived from single-and multiunit recordings in the decerebrate, unanesthetized cat. This approach provided details about the arrangement of the corticocuneate pathway that were not available from previous studies with anterograde degeneration methods.

On the basis of cytoarchitectonic and connectional features, a number of subdivisions are identified in the cuneate nucleus, each of which is associated with characteristic functional properties. In agreement with previous studies, it is found that a large portion of the cuneate nucleus, the middle dorsal part (MCd), is exclusively devoted to the representation of cutaneous receptive fields on the digits. This “core” region contains more thalamic projecting neurons than any other subdivision of the cuneate nucleus. A topographic arrangement also exists in the subdivisions of the rostral cuneate and of the nuclear region ventral to MCd, although in these, receptive fields are larger and predominantly, but not exclusively, related to deep receptors and involve the arm, shoulder, and trunk.

Observations on corticocuneate projections were based on injections, mainly focused on functional subdivisions of the primary somatosensory cortex (SI) as described by McKenna et al (1981). Although cortical projections are mainly to cuneate regions other than its core, a significant proportion of fibers from the region of SI where the digits are represented (particularly area 3b) do project to the MCd region of the cuneate nucleus. Similarly, nuclear areas associated with receptive fields on the arm and trunk are labeled after injection in SI arm and trunk regions, respectively. Thus, a close topographic relationship appears to exist between the somatosensory cortex and cuneate regions related to the same body representation, although nuclear regions in which receptive fields on the neck area are represented receive very sparse or no detectable cortical projections even when the injection of the tracer involves the entire sensorimotor cortex. The topographic arrangement of SI projections upon the cuneate nucleus suggests that a similar pattern exists in both structures with regard to the relative representations of distal versus proximal and deep versus cutaneous receptive fields (e.g., “core” vs. “shell” organization), and that cuneate regions preferentially related to either of these classes of receptive fields receive direct connections from the corresponding regions in SI.

A comparison of the results from cats with tracer injections in areas 4 and 3b reveals that the projections from the former is denser than that arising from the latter and that their territories of termination largely overlap in the ventral portions of the cuneate nucleus. However, cortical projections to MCd may be derived from the somatosensory cortex with no contribution from area 4. The demonstration of the relative selectivity of cortical projections from different cytoarchitectonic and functional cortical areas to cuneate regions identified here provides a structural basis for the elucidation of the physiological and behavioral observations, particularly on cortical modulation of somatosensory transmission during movements.     

Actin-filled nuclear invaginations indicate degree of cell de-differentiation

For years the existence of nuclear actin has been heavily debated, but recent data have clearly demonstrated that actin, as well as actin-binding proteins (ABPs), are located in the nucleus. We examined live EGFP-actin-expressing cells using confocal microscopy and saw the presence of structures strongly resembling actin filaments in the nuclei of MDA-MB-231 human mammary epithelial tumor cells. Many nuclei had more than one of these filamentous structures, some of which appeared to cross the entire nucleus. Extensive analysis, including fluorescence recovery after photobleaching (FRAP), showed that all EGFP-actin in the nucleus is monomeric (G-actin) rather than filamentous (F-actin) and that the apparent filaments seen in the nucleus are invaginations of cytoplasmic monomeric actin. Immunolocalization of nuclear pore complex proteins shows that similar invaginations are seen in cells that are not overexpressing EGFP-actin. To determine whether there is a correlation between increased levels of invagination in the cell nuclei and the state of de-differentiation of the cell, we examined a variety of cell types, including live Xenopus embryonic cells. Cells that were highly de-differentiated, or cancerous, had an increased incidence of invagination, while cells that were differentiated had few nuclear invaginations. The nuclei of embryonic cells that were not yet differentiated underwent multiple shape changes throughout interphase, and demonstrated numerous transient invaginations of varying sizes and shapes. Although the function of these actin-filled invaginations remains speculative, their presence correlates with cells that have increased levels of nuclear activity.     

Fluorescent in situ hybridization of the telomere repeat sequence in hamster sperm nuclear structures

The flat, hooked-shaped architecture of the hamster sperm nucleus makes this an excellent model for in situ hybridization studies of the three dimensional structure of the genome. We have examined the structure of the telomere repeat sequence (TTAGGG)_n with respect to the various nuclear structures present in hamster spermatozoa, using fluorescent in situ hybridization. In fully condensed, mature sperm nuclei, the telomere sequences appeared as discrete spots of various sizes interspersed throughout the volume of the nuclei. While the pattern of these signals was non-random, it varied significantly in different nuclei. These discrete telomere foci were seen to gradually lengthen into linear, beaded signals as sperm nuclei were decondensed, in vitro, and were not associated with the nuclear annulus. We also examined the relationship of telomeres to the sperm nuclear matrix, a residual nuclear structure that retains the original size and shape of the nucleus. In these structures the DNA extends beyond the perimeter of the nucleus to form a halo around it, representing the arrangement of the chromosomal DNA into loop domains attached at their bases to the nuclear matrix. Telomere signals in these structures were also linear and equal in length to those of the decondensed nuclei, and each signal represented part of a single DNA loop domain. The telomeres were attached at one end to the nuclear matrix and extended into the halo. Sperm nuclear matrices treated with Eco RI retained the telomere signals. These data support sperm DNA packaging models in which DNA is coiled into discrete foci, rather than spread out linearly along the length of the sperm nucleus.     

Eliminating the synthesis of mature lamin A reduces disease phenotypes in mice carrying a Hutchinson-Gilford progeria syndrome allele

Hutchinson-Gilford progeria syndrome is caused by the synthesis of a mutant form of prelamin A, which is generally called progerin. Progerin is targeted to the nuclear rim, where it interferes with the integrity of the nuclear lamina, causes misshapen cell nuclei, and leads to multiple aging-like disease phenotypes. We created a gene-targeted allele yielding exclusively progerin (Lmna HG) and found that heterozygous mice (Lmna HG/+) exhibit many phenotypes of progeria. In this study, we tested the hypothesis that the phenotypes elicited by the Lmna HG allele might be modulated by compositional changes in the nuclear lamina. To explore this hypothesis, we bred mice harboring one Lmna HG allele and one Lmna LCO allele (a mutant allele that produces lamin C but no lamin A). We then compared the phenotypes of Lmna HG/LCO mice (which produce progerin and lamin C) with littermate Lmna HG/+ mice (which produce lamin A, lamin C, and progerin). Lmna HG/LCO mice exhibited improved HG/LCO fibroblasts had fewer misshapen nuclei than Lmna HG/+ fibroblasts (p < 0.0001). A likely explanation for these differences was uncovered; the amount of progerin in Lmna HG/LCO fibroblasts and tissues was lower than in Lmna HG/+ fibroblasts and tissues. These studies suggest that compositional changes in the nuclear lamina can influence both the steady-state levels of progerin and the severity of progeria-like disease phenotypes.     

DNA synthesis in the fertilizing hamster sperm nucleus: sperm template availability and egg cytoplasmic control

To assess the role of the availability of sperm nuclear templates in the regulation of DNA synthesis, we correlated the morphological status of the fertilizing hamster sperm nucleus with its ability to synthesize DNA after in vivo and in vitro fertilization. Fertilized hamster eggs were incubated in 3H-thymidine for varying periods before autoradiography. None of the decondensed sperm nuclei nor early (Stage I) male pronuclei present after in vivo or in vitro fertilization showed incorporation of label, even in polyspermic eggs in which more advanced pronuclei were labeled. In contrast, medium-to-large pronuclei (mature Stage II pronuclei) consistently incorporated 3H-thymidine. To investigate the contribution of egg cytoplasmic factors to the regulation of DNA synthesis, we examined the timing of DNA synthesis by microinjected sperm nuclei in eggs in which sperm nuclear decondensation and male pronucleus formation were accelerated experimentally by manipulation of sperm nuclear disulfide bond content. Although sperm nuclei with few or no disulfide bonds decondense and form male pronuclei faster than nuclei rich in disulfide bonds, the onset of DNA synthesis was not advanced. We conclude the the fertilizing sperm nucleus does not become available to serve as a template for DNA synthesis until it has developed into a mature Stage II pronucleus, and that, as with decondensation and pronucleus formation, DNA synthesis also depends upon egg cytoplasmic factors.     

Cytological Events during Zygote Formation of the Fern Ceratopteris thalictroides

The cytological events, including nuclear fusion, digestion of male organelles and rebuilding of the plasmalemma and cell wall, during zygote formation of the fern Ceratopteris thalictroides (L.) Brongn. are described based on the observations of transmission electron microscopy. When the spermatozoid enters the egg and contacts the cytoplasm, the male chromatin relaxes continually. The microtubular ribbon (MTr) is separated from the male nucleus and then an envelope reappears around the male nucleus. During nuclear fusion, the egg nucleus becomes highly irregular and extends some nuclear protrusions. It is proposed that the protrusions fuse with the male nucleus actively. After nuclear fusion the irregular zygotic nucleus contracts gradually. It becomes spherical before the zygote divides. The male chromatin is identifiable as fibrous structure in the zygotic nucleus in the beginning, but it gradually becomes diffused completely. The male organelles, including the MTr, multilayered structure, flagella and the male mitochondria are finally digested in the zygotic cytoplasm. Finally a new plasmalemma and cell wall are formed outside the protoplast. The organelles in the zygote are rearranged, which produces a horizontal polarity zygote. The zygote divides with an oblique-vertical cell plate facing the apical notch of the gametophyte.     

Copepod development rates in relation to genome size and 18S rDNA copy number.

It is known that body sizes and temperature-independent developmental durations within two genera of calanoid Copepoda (Crustacea) are positively related to nuclear DNA contents of their somatic nuclei. Evidently because of the constraint of similar cell numbers among the species, (nucleotypic) effects of nucleus size on cell size and on cell-level processes are expressed at the whole-organism level. Here, we show that developmental durations of eight species of five genera are also negatively related to their greatly differing numbers of 18S rRNA genes per unit DNA. We propose that levels of rDNA iteration among copepods have been controlled by natural selection to regulate ribosome concentrations, therefore protein production and development rates, independently of the large variations in genome sizes, which are in turn adapted to regulating cell and therefore body sizes.     

Development of sex differences in the principal nucleus of the bed nucleus of the stria terminalis of mice: role of Bax-dependent cell death

Neuron number in the principal nucleus of the bed nucleus of the stria terminalis (BNSTp) is greater in adult male mice than in females. Deletion of the proapoptotic gene, Bax, increases the number of BNSTp cells in adulthood and eliminates the sex difference in cell number. Here, we map the ontogeny of sex differences in nuclear volume and cell number in the BNSTp of neonatal mice, and evaluate the role of cell death in the development of these differences. We find that BNSTp volume and cell number do not differ between male and female wild-type mice on postnatal days P3, P5, or P7. Sex differences emerge after the first postnatal week and both measures are significantly greater in males than in females on P9 and P11. Cell death, assessed by TUNEL staining, was observed in the BNSTp of both sexes from P1-P8. Females had more TUNEL-positive cells than males from approximately P3-P6, with the maximum number of dying cells observed on P5/P6. To test whether the Bax gene is required for sexually dimorphic cell death in the BNSTp, TUNEL cells were counted on P6 in Bax -/- mice and their Bax +/+ siblings. Bax gene deletion nearly abolished TUNEL-positive cells in the BNSTp of both sexes. Together, these findings support the interpretation that the sex difference in BNSTp cell number seen in adulthood is due to Bax-dependent, sexually dimorphic cell death during the first week of life.