Sex Differences In Cell Number Research Articles

New cells added to the preoptic area during sex change in the common clownfish Amphiprion ocellaris

Sex differences in cell number in the preoptic area of the hypothalamus (POA) are documented across all major vertebrate lineages and contribute to differential regulation of the hypothalamic-pituitary-gonad axis and reproductive behavior between the sexes. Sex-changing fishes provide a unique opportunity to study mechanisms underlying sexual differentiation of the POA. In anemonefish (clownfish), which change sex from male to female, females have approximately twice the number of medium-sized cells in the anterior POA compared to males. This sex difference transitions from male-like to female-like during sex change. However, it is not known how this sex difference in POA cell number is established. This study tests the hypothesis that new cell addition plays a role. We initiated adult male-to-female sex change in 30 anemonefish (Amphiprion ocellaris) and administered BrdU to label new cells added to the POA at regular intervals throughout sex change. Sex-changing fish added more new cells to the anterior POA than non-changing fish, supporting the hypothesis. The observed effects could be accounted for by differences in POA volume, but they are also consistent with a steady trickle of new cells being gradually accumulated in the anterior POA before vitellogenic oocytes develop in the gonads. These results provide insight into the unique characteristics of protandrous sex change in anemonefish relative to other modes of sex change, and support the potential for future research in sex-changing fishes to provide a richer understanding of the mechanisms for sexual differentiation of the brain.

Epigenetic Mechanisms of Brain Sexual Differentiation.

Across vertebrate species, gonadal hormones coordinate physiology with behavior to facilitate social interactions essential for reproduction and survival. In adulthood, these hormones activate neural circuits that regulate behaviors presenting differently in females and males, such as parenting and territorial aggression. Yet long before sex-typical behaviors emerge at puberty, transient hormone production during sensitive periods of neurodevelopment establish the circuits upon which adult hormones act. How transitory waves of early-life hormone signaling exert lasting effects on the brain remains a central question. Here we discuss how perinatal estradiol signaling organizes cellular and molecular sex differences in the rodent brain. We review classic anatomic studies revealing sex differences in cell number, volume, and neuronal projections, and consider how single-cell sequencing methods enable distinction between sex-biased cell-type abundance and gene expression. Finally, we highlight the recent discovery of a gene regulatory program activated by estrogen receptor α (ERα) following the perinatal hormone surge. A subset of this program displays sustained sex-biased gene expression and chromatin accessibility throughout the postnatal sensitive period, demonstrating a bona fide epigenetic mechanism. We propose that ERα-expressing neurons throughout the social behavior network use similar gene regulatory programs to coordinate brain sexual differentiation.

Sex Differences and the Roles of Sex Steroids in Apoptosis of Sexually Dimorphic Nuclei of the Preoptic Area in Postnatal Rats

The brain contains several sexually dimorphic nuclei that exhibit sex differences with respect to cell number. It is likely that the control of cell number by apoptotic cell death in the developing brain contributes to creating sex differences in cell number in sexually dimorphic nuclei, although the mechanisms responsible for this have not been determined completely. The milieu of sex steroids in the developing brain affects sexual differentiation in the brain. The preoptic region of rats has two sexually dimorphic nuclei. The sexually dimorphic nucleus of the preoptic area (SDN-POA) has more neurones in males, whereas the anteroventral periventricular nucleus (AVPV) has a higher cell density in females. Sex differences in apoptotic cell number arise in the SDN-POA and AVPV of rats in the early postnatal period, and an inverse correlation exists between sex differences in apoptotic cell number and the number of living cells in the mature period. The SDN-POA of postnatal male rats exhibits a higher expression of anti-apoptotic Bcl-2 and lower expression of pro-apoptotic Bax compared to that in females and, as a potential result, apoptotic cell death via caspase-3 activation more frequently occurs in the SDN-POA of females. The patterns of expression of Bcl-2 and Bax in the SDN-POA of postnatal female rats are changed to male-typical ones by treatment with oestrogen, which is normally synthesised from testicular androgen and affects the developing brain in males. In the AVPV of postnatal rats, apoptotic regulation also differs between the sexes, although Bcl-2 expression is increased and Bax expression and caspase-3 activity are decreased in females. The mechanisms of apoptosis possibly contributing to the creation of sex differences in cell number and the roles of sex steroids in apoptosis are discussed.

Control of Cell Number in the Sexually Dimorphic Brain and Spinal Cord

The hormonal control of cell death is currently the best-established mechanism for creating sex differences in cell number in the brain and spinal cord. For example, males have more cells than do females in the principal nucleus of the bed nucleus of the stria terminalis (BNSTp) and spinal nucleus of the bulbocavernosus (SNB), whereas females have a cell number advantage in the anteroventral periventricular nucleus (AVPV). In each case, the difference in cell number in adulthood correlates with a sex difference in the number of dying cells at some point in development. Mice with over- or under-expression of cell death genes have been used to test more directly the contribution of cell death to neural sex differences, to identify molecular mechanisms involved, and to determine the behavioural consequences of suppressing developmental cell death. Bax is a pro-death gene of the Bcl-2 family that is singularly important for apoptosis in neural development. In mice lacking bax, the number of cells in the BNSTp, SNB and AVPV are significantly increased, and sex differences in total cell number in each of these regions are eliminated. Cells rescued by bax gene deletion in the BNSTp express markers of differentiated neurones and the androgen receptor. On the other hand, sex differences in other phenotypically identified populations, such as vasopressin-expressing neurones in the BNSTp or dopaminergic neurones in AVPV, are not affected by either bax deletion or bcl-2 over-expression. Possible mechanisms by which testosterone may regulate cell death in the nervous system are discussed, as are the behavioural effects of eliminating sex differences in neuronal cell number.

Sex differences in NeuN- and androgen receptor–positive cells in the bed nucleus of the stria terminalis are due to Bax-dependent cell death

The principal nucleus of the bed nucleus of the stria terminalis (BNSTp) is larger in males than in females of several species. We previously demonstrated that in mice lacking the pro-death gene, bax, total BNSTp cell number is increased and sex differences in cell number are eliminated. This suggests that Bax-dependent cell death underlies sexual differentiation of the BNSTp. However, it is not known what cells in the BNSTp are affected by bax deletion. Here we used immunohistochemistry and stereological techniques to quantify phenotypically-identified cells in the BNSTp of adult male and female bax −/− and bax +/+ mice. Sections were thionin-stained, or double-labeled for antigen expressed in neuronal nuclei (NeuN) and glial fibrillary acidic protein (GFAP) to identify mature neurons and astrocytes, respectively; an additional series was labeled for androgen receptor (AR). As previously demonstrated, sex differences in BNSTp area and overall cell number were seen in wild-type mice, but absent in bax −/− animals. In addition, sex differences (favoring males) were present in the number of NeuN+ and AR+ cells in wild-type mice. Bax gene deletion significantly increased the number of NeuN+ and AR+ cells and reduced or eliminated the sex differences in these cell types. The number of astrocytes in the BNSTp was not sexually dimorphic, nor significantly affected by bax gene status, although there was a trend for more GFAP+ cells in bax −/− mice. Overall brain weight was also greater in bax −/− animals compared with controls. We conclude that the sex differences in neuron and AR+ cell number are due at least in part to Bax-mediated cell death. Increased NeuN+ and AR+ cell number in bax −/− mice suggests that supernumerary cells in bax knockouts differentiate similarly to those in wild-type mice, and retain the capacity to respond to androgens.

Deletion of the Bax gene disrupts sexual behavior and modestly impairs motor function in mice

Cell death is a nearly ubiquitous feature of the developing nervous system, and differential death in males and females contributes to several well studied sex differences in neuron number. Nonetheless, the functional importance of neuronal cell death has been subjected to few direct tests. Bax, a pro-apoptotic protein, is required for cell death in many neural regions. Deletion of the Bax gene in mice increases neuron number in several areas and eliminates sex differences in cell number in the brain and spinal cord. Here, sexual and motor behaviors were examined in Bax-/- mice and their wild-type siblings to test the functional consequences of preventing Bax-dependent cell death. Animals were gonadectomized in adulthood and provided with ovarian hormones or with testosterone for tests of feminine and masculine sexual behaviors, respectively. Wild-type mice exhibited a sex difference in feminine sexual behavior, with high lordosis scores in females and low scores in males. This sex difference was eliminated by Bax deletion, with very low receptivity exhibited by both male and female Bax-/- mice. Masculine sexual behavior was not sexually dimorphic among wild-type mice, but mounts and pelvic thrusts were nearly eliminated in Bax-/- mice of both sexes. Motor strength and performance at low speeds on a RotaRod apparatus did not differ by sex or Bax gene status. However, Bax-/- animals exhibited impairments on the RotaRod at higher speeds. Thus, developmental cell death may be required for masculine and feminine sexual behaviors and the fine tuning of motor coordination.

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.

Sex differences in the location of immunochemically defined cell populations in the mouse preoptic area/anterior hypothalamus

The preoptic area/anterior hypothalamus (POA/AH) is sexually dimorphic in many vertebrates. We have defined specific cell populations within the POA/AH using immunocytochemical markers for estrogen receptor β (ERβ) and the R1 subunit of the GABA B receptor (GABA BR1). Our previous finding of sex differences in cell migration in this region in embryonic day 15 mice led us to examine sex differences in the location or size of chemically identified cell groups. At embryonic day 17 (E17), cells containing immunoreactive (ir) ERβ in females were located more dorsal and lateral than those in males. In contrast to this positional sex difference seen at E17, ERβ expression at P0 and adulthood showed a sex difference in cell number and area of immunoreactivity with a higher expression of ERβ in males than females. Furthermore, in animals that were genetically deprived of gonadal and adrenal hormones by virtue of a disrupted gene coding for steroidogenic factor 1, cells containing ir ERβ followed a female phenotype for location at E17 and a female phenotype for number of ir cells at P0 regardless of genetic sex, suggesting that circulating hormones may be influencing cell position in the POA/AH. A second phenotypically identified cell group containing ir GABA BR1 also had a sex difference in cell positions at E17. Females expressed GABA BR1 in cells with a more dorsal position than in males. These results provide support for the suggestion that sex differences in cellular organization in the developing hypothalamus arise from sex differences in cell migration.

Sex differences in the magnocellular subdivision of the medial preoptic nucleus in Syrian hamsters

Exposure to pheromonal cues initiates male mating behavior. Pheromones are processed within a pathway that converges on the magnocellular subdivision of the medial preoptic nucleus. Lesions of this area eliminate male copulatory behavior, but do not affect anogenital investigation. Exposure to pheromones stimulates cells of the magnocellular subdivision of the medial preoptic nucleus in a sex-specific manner. In this study, we hypothesize that sex differences in cell number may underlie sex differences in pheromone-induced neural stimulation. The current study used unbiased stereological methods to identify sexual dimorphisms in the magnocellular subdivision of the medial preoptic nucleus. Sex differences were found in the number and density of neurons, but not in overall volume or neuron volume. Consequently, the total volume is not sexually differentiated because neurons are more densely packed within the male magnocellular subdivision of the medial preoptic nucleus. These results support the hypothesis that additional neurons in the magnocellular subdivision of the medial preoptic nucleus are critical for the expression of male copulatory behaviors in adulthood. Furthermore, they suggest that sexual differentiation of the magnocellular subdivision of the medial preoptic nucleus is an important process that forms the anatomical basis for sex-specific behavioral responses to pheromonal stimulation.

Motoneurons innervating guinea pig perineal muscles are sexually dimorphic in size but not number

Sexual differentiation occurs prenatally in guinea pigs but extends into the postnatal period in rats. Steroids affect the development of two motoneuron nuclei of the rat lumbar spinal cord that innervate sexually dimorphic perineal muscles. The spinal nucleus of the bulbocavernosus (SNB) innervates the bulbocavernosus (BC) and levator ani (LA) muscles while the dorsolateral nucleus (DLN) innervates the ischiocavernosus (IC). In male rats, perinatal testosterone prevents degeneration of these muscles and results in a sex difference in both motoneuron size and number in adulthood. For comparative purposes, we examined the guinea pig motoneurons innervating these muscles, as well as those innervating the retractor penis (RP) and retractor clitoris (RC), muscles that have no counterpart in rats. Injections of horseradish peroxidase localized the BC LA and IC motoneurons of guinea pigs to discrete columns in spinal levels L6 and S1, with the BC LA motoneurons occupying a more medial position. The RP RC motoneurons were found in L5. Motoneuronal soma area was larger in males in all examined motor pools, as was nuclear area of BC LA and IC motoneurons. Although raw counts suggested a sex difference in cell number in the motor columns containing BC LA and IC motoneurons, either of two different correction procedures for split nuclei error eliminated the sex difference in cell number, emphasizing the importance of such corrections when comparing neurons of different size.