Male mice have a cluster of calbindin-ir cells in the POA that mirrors a similar cell group in the SDN-POA of rats [28
]. Although previous studies uniformly report that the volume of this cluster is larger in males, some controversy exists about whether this is due to a sex difference in the number of calbindin-ir cells. Using a uniform contour superimposed in a consistent manner over sections of the POA in males and females, we confirm that the CALB-SDN contains about three times as many cells in male than in female mice. In addition, a single injection of EB on the day of birth completely masculinizes cell number within the CALB-SDN of females.
These findings support and extend the report of Orikasa and Sakuma [31
] that male mice have more cells in the CALB-SDN than do females and that five daily injections of EB between postnatal days 1 and 5 masculinized cell number in females. In contrast, cell counts in the CALB-SDN were not affected by adult gonadectomy or hormone replacement [31
]. Similarly, in rats, volume of the CALB-SDN was reduced in males by gonadectomy on the day of birth and increased in females treated neonatally with either testosterone proionate or EB [27
]. This indicates that sexual differentiation of the CALB-SDN is due to organizational effects of estrogenic steroids. However, there may also be a role for androgens, at least in mice, because disruption of the androgen receptor results in an intermediate phenotype (that is, volume of the CALB-SDN is not significantly different from females or males), as does the neonatal treatment of female mice with the non-aromatizable androgen dihydrotestosterone [28
With the use of grid counts we also examined the number of calbindin-ir cells surrounding the main cluster. Males had more calbindin-ir cells not only within the CALB-SDN, but also in adjacent regions. This supports the interpretation that total calbindin-ir cell number in the POA is sexually dimorphic; we did not find evidence for an equal number of cells that are more scattered in females. A limitation of this conclusion is that our grid counts examined cells distributed in two dimensions (ventral to dorsal and medial to lateral) and only within the confines of the grid. We cannot rule out the possibility that females have more calbindin-ir cells scattered rostrocaudally from the central cluster or beyond the perimeter or our grid, although a simple visual inspection did not suggest either possibility.
The sex difference in cell number in the CALB-SDN mirrors the sex difference in total cell number in the rat SDN-POA which, as described above, is thought to be due primarily to estrogen-regulated developmental cell death [11
]. We therefore hypothesized that cell death was the mechanism underlying sexual differentiation of the CALB-SDN, and tested this by comparing wild-type and Bax
knockout mice. Cell number is increased in many brain regions of Bax
knockouts and several sex differences in the brain and behavior are eliminated in Bax
-/- mice [25
]. Surprisingly, however, Bax
gene deletion did not increase the number of calbindin-ir cells in the CALB-SDN or surrounding regions of either sex. Thus, our results do not support a role for cell death in regulating either total cell number or the sex difference in cell number in the CALB-SDN.
The calbindin cluster is often taken as representative of the SDN-POA as a whole. The current findings suggest that either the role of cell death in the SDN-POA should be reconsidered, or that there are different mechanisms underlying the differentiation of calbindin-ir cells and other cells in the nucleus. Several caveats should be considered, however. For example, it is possible that the sex difference in the number of calbindin-ir cells is in fact due to cell death, but it is Bax-independent cell death. This seems somewhat unlikely given the crucial role of Bax and Bcl-2 in controlling cell death throughout the developing brain and the fact that Bax deletion increased total cell density in the cluster region. In addition, apoptotic cells, as identified by activated caspase-3 immunoreactivity, are essentially eliminated in the medial preoptic area (as well as most other brain areas) of Bax knockout mice postnatally (Ahern T, Carr A and Forger NG, unpublished results), suggesting negligible Bax-independent cell death in this region. If cell death contributes to calbindin-ir cell number in the POA, it would have to be both Bax and caspase-3 independent.
A second possible limitation to the conclusion that cell death does not contribute to development of the CALB-SDN is that presumptive calbindin-ir cells may be rescued from cell death by Bax
deletion, but fail to make calbindin protein. Supernumerary cells in the BNSTp and anteroventral periventricular nucleus of Bax
knockout mice express proteins such as vasopressin, NeuN, the androgen receptor (AR) and kisspeptin [36
], indicating that they differentiate normally to the extent of expressing at least some relevant markers. However, the 'extra' cells in Bax
knockout mice may not always differentiate normally (see, for example, [42
]). The inability of cells rescued by Bax
deletion to specifically express calbindin seems unlikely here, however, because we found a significant increase in the number of calbindin-ir cells in the BNSTp of Bax
Taken together, these findings indicate that cell death is not the only mechanism underlying sex differences in cell number in the SDN-POA, at least not the sex difference in calbindin-ir cell number. Differential neurogenesis, migration, or phenotypic differentiation are other possible mechanisms. Previous studies examining the birthdate of cells comprising the SDN-POA of rats have not found evidence for a sex difference in neurogenesis [11
]. A sex difference in the migration or aggregation of cells into the nucleus is possible, especially in light of work demonstrating effects of sex and estradiol on cell migration in embryonic slice cultures of the POA [45
]. We note, however, that if differences in the migration or aggregation of cells account for sexual differentiation of the CALB-SDN, calbindin-ir cells in females must end up quite a distance away from the cluster seen in males (that is, beyond the boundaries of the grid used for counts here). A more likely mechanism may be the differentiation of neurochemical phenotype. That is, more cells may be induced to differentiate into calbindin-expressing cells in males, presumably in response to perinatal exposure to testosterone and its metabolites. Similarly, other sex differences in the medial POA of rats depend on the differentiation of (morphological) phenotype, for example, the morphology of astrocytes is more complex and the density of dendritic spines is greater in the POA of male than in female rats [47
In addition to the sex difference in the CALB-SDN, we also found a sex difference in the number of calbindin-ir cells in the BNSTp. The calbindin-ir cells in the BNSTp have not received much attention although they comprised the largest and most striking accumulation of calbindin-positive cells in our material. In the only other report on these cells that we are aware of, and consistent with the present findings, Büdefeld et al.
] reported more calbindin immunoreactivity in the encapsulated portion of the BNSTp of male than of female mice. This sex difference was seen in animals that had been gonadectomized and treated with testosterone propionate in adulthood indicating that, similar to the sex difference in the CALB-SDN, it is independent of adult hormonal state [29
Preventing cell death by Bax
gene deletion increases the number of Nissl-stained cells and AR-expressing cells in the BNSTp and eliminates sex differences in total and AR cell number [25
]. Deleting Bax
also increased the number of calbindin-ir cells in the BNSTp here. However, the increase was roughly equivalent (approximately 1,000 cells) in males and females so the sex difference was not eliminated. Thus, although cell death determines the number of potential calbindin-ir cells in both sexes, a second mechanism must be responsible for the sex difference. As for the CALB-SDN, we suggest that this second mechanism is the differentiation of neurochemical phenotype (that is, more cells become calbindin-expressing neurons in males, under the control of hormones or sex chromosomes).
The fact that Bax
deletion increased calbindin-ir cell number in the BNSTp but not the CALB-SDN raises the question of what underlies this regional difference. Although calbindin has been used as a 'marker' of the SDN-POA it is also a calcium binding protein associated with neuroprotection (see, for example, [49
]). In rats, calbindin is expressed at higher levels in males in the preoptic area/mediobasal hypothalamus generally, and the SDN-POA specifically, prior to the period of sexually dimorphic cell death [27
]. It is therefore possible that perinatal testosterone directs more cells to become calbindin-expressing in males and that those cells with calbindin protein are relatively protected from cell death. That is, Bax
gene deletion may have had no effect on the number of cells in the CALB-SDN because calbindin-expressing cells are normally spared from pruning in wild-type animals. To our knowledge, it is not known if the ontogeny of calbindin expression differs between the CALB-SDN and BNSTp, but an earlier emergence of calbindin in the CALB-SDN could be one possible explanation for why Bax
deletion influences calbindin cell number differently in the two nuclei.