In the present study, we found that exogenous oestradiol treatment promoted cell proliferation and survival in the neonatal female but not the male hippocampus, whereas antagonizing endogenous oestradiol synthesis or action reduced cell proliferation in the male but not in the female hippocampus. These results confirm and extend our previous report describing a sex difference and oestradiol-induced increase in the number of new cells in the neonatal hippocampus  by confirming a sex difference in cell genesis that is regulated by oestradiol. Our previous report included evidence of a male biased sex difference and oestradiol-induced increase in BrdU+/glial fibrillary acid (GFAP)+ expressing cells. We also quantified the number of NeuN+ cells on PN4, but did not co-label them with BrdU as there was insufficient time for neurons born on PN0 to differentiate. We found more NeuN+ cells in males, but there was no significant increase following oestradiol treatment at PN0-1. This is, again, most probably due to insufficient time for neurons born on PN0 to differentiate into neurons. Therefore our previous study did not assess neurogenesis per se, as opposed to the later developmental stage of PN21 used here. Moreover, many GFAP expressing cells ultimately become neurons  and it is possible the oestradiol-induced increase we observed in BrdU+/GFAP+ cells was a precursor to the later increase in BrdU+/NeuN+ cells seen here. The current analysis is a more accurate depiction of the fate of cells labelled at birth with BrdU under the influence of oestradiol, whereas our previous results depict the fate of new cells within a few days of birth.
There are two mechanisms by which an increase in new cell number can occur; (1) an increased rate of proliferation; and/or (2) a decreased rate of cell death. In our previous study we observed a significant increase in the number of new cells within 24 h of BrdU injection in males versus females and in females treated with oestradiol versus vehicle treated females. In the current study the same pattern was observed at the even shorter post-BrdU injection time point of 8 h. The half-life of BrdU is approximately 2 h  whereas the cell cycle requires approximately 24 h to complete . Therefore, differences in the number of BrdU+ cells within 24 h of injection are generally interpreted as differences in the rate of cell proliferation and not cell death . Our observation of more BrdU+ cells in males and oestradiol-treated females at both 8 h and 24 h post-BrdU injection is, therefore, most consistent with a hormonally mediated sex difference in cell proliferation. However, given that many new cells die shortly after being born and would not be detected here, a potential contribution of cell death to the sex differences observed cannot be entirely ruled out.
While BrdU labelling offers many advantages, such as long-term tracking of cell fate, there are also limitations associated with potential toxicity and non-specific labelling . In contrast, Ki-67 is an endogenous protein that does not have any adverse effects on living cells and is expressed in all phases of the cell cycle except the resting phase and a short period at the beginning of the G1 phase [39–41]. Thus, Ki-67 is not present in quiescent and terminally differentiated cells and increased numbers of Ki-67 expressing cells is consistent with increased proliferation (but see ). In order to determine if sex differences or hormonal modulation of cell proliferation persisted outside of the early neonatal period we quantified the number of Ki-67 expressing cells in the hippocampus of 3-week-old animals and found males and neonatally oestradiol-treated females had more Ki-67+ cells than control females. This observation suggests a higher rate of cell proliferation was organized during the neonatal sensitive period. A precedent for the sexual differentiation of neurogenesis is evident in studies reporting differential sensitivity of adult males and females to exogenous oestradiol treatment or rates of cell birth and death in the DG. In the adult female hippocampus, oestradiol stimulates cell proliferation , enhances cell survival  and increases dendritic spine synapse density [45–48]. In contrast, the adult male hippocampus is insensitive to the spinogenesis or cell genesis inducing effects of oestradiol treatment [44, 49, 50]. Oestradiol effects are mediated via binding to the two oestrogen receptor isoforms, ERα and ERβ [51, 52]. Both isoforms are distributed throughout the brain, including the hippocampus, and both ERs colocalize with the proliferative marker Ki-67 in the adult hippocampus [50, 53, 54], although, there is a region specific variation in their distribution [54–57]. Moreover, in the adult hippocampus, there are relatively low levels ERα, which is in contrast to elevated levels of ERα that occur during early postnatal development [58–60]. Nonetheless, it is unclear which receptor isoform is regulating the oestradiol's effects on cell proliferation in the developing hippocampus. Both the short-term increase in the number of BrdU cells and the higher density of Ki-67+ cells in oestradiol treated females indicate a change in proliferative rates of progenitor cells induced by oestradiol and this may be due to a change in the duration of the progenitor cell cycle. This was determined to be the case in neocortical neurogenesis . Oestradiol recruits cells into the S-phase from either G1 or G0 phase, which effectively shortens the G-phase, and results in an increased rate of proliferation of dividing progenitor cells .
Both the amount of endogenous oestradiol and aromatase activity in the developing hippocampus are extremely low compared to the hypothalamus and do not appear to be sexually dimorphic , suggesting that hippocampal sensitivity to oestradiol is high and differs between the sexes, at least as indexed by hippocampal cell genesis. However, because oestradiol, the ER antagonist and the aromatase inhibitor were all administered systemically, it is possible the effects were not mediated directly at the hippocampus but, instead, were secondary to changes in other brain regions projecting to the hippocampus. Cholinergic neurons of the medial septum/diagonal band of Broca are essential for oestradiol-induced spinogenesis in adult CA1 hippocampus  and cholinergic input modulates maturation and integration of adult born DG granule cells . Gonadally intact males release more acetylcholine into the hippocampus than females during locomotor tasks and this sex difference is organized by oestradiol during development [65, 66]. The cholinergic system matures relatively early and more new septal cholinergic neurons are born in males during a brief period of gestation but the sex difference does not persist into adulthood . Nonetheless, it is possible that the effects observed here are the results of oestradiol-induced acetylcholine release into the neonatal hippocampus during the early postnatal period. It is also possible that oestradiol is acting outside the central nervous system. In adults, systemic oestradiol alters the arterial cerebral blood flow in females, but not in males [68–71], via an interaction with nitric oxide [72, 73]. Both ERα and ERβ, as well as the transmembrane G protein-coupled receptor, GPR30, have been identified in blood vessels [74, 75] and have been implicated in the rapid vasodilator effects of oestradiol . One of the most powerful stimulators of adult neurogenesis is exercise [77, 78]; an effect believed to be at least partly due to enhanced blood flow and increased delivery of growth factors. Lastly, activation of the stress axis has negative effects on adult neurogenesis and the injection of BrdU and steroidal agents to neonates is undoubtedly stressful. However, given that the number of injections was carefully controlled for across groups, sex differences in stress responding can not entirely explain the current results.
Neurogenesis in the adult hippocampus is restricted to the proliferative zone of DG, with the majority of the new cells becoming granule neurons [79, 80]. A notable difference in the profile of neurogenesis in the immature brain is the presence of ongoing proliferation in CA1 and CA3 of Ammon's horn. The colocalization of BrdU with NeuN in these areas indicates that the cells born early postnatally do become neurons but whether they become pyramidal neurons or interneurons is unknown. When, in development, this source of new cells is lost and neurogenesis becomes restricted to the DG is also unknown. An additional unknown is the ultimate role of these enduring neurons in the adult hippocampal function. We observed almost twice as many new cells being born in the neonatal male hippocampus compared to the female. However, when the overall size of the hippocampus is compared in males and females, either developmentally or in adulthood, the sex difference, while biased towards males, is of the order of 10%-12% [15, 21]. The magnitude of the sex difference in new cells was just as strong at 3 weeks of age as when they were just a few days old. Therefore, it does not appear that these cells contribute significantly to the hippocampal volume. The early postnatal period is a time of olfactory imprinting and somatosensory stimulation from the dam. The intensity of maternal licking and grooming is greater toward male than female pups  and both the nature and function of olfactory learning at this time is likely to be different between the sexes. Whether the role of new neurons born during this period is related to olfactory or sensory learning remains to be determined.