Article Figures & Data
Figures
- Figure 1.
Pubertally born cells were activated by estradiol and P treatment. A, Representative images of a pubertally born cell that was also Fos-ir after a hormone-induced LH surge in adulthood. Images are a maximum intensity projection of a Z-stack (0.5-μm step) captured using the MBF Bioscience Image Stack Module. Scale bar, 10 μm. An orthogonal view (right) through the middle of the cell confirms colocalization; red arrow, x plane; green arrow, y plane; and blue arrow, z plane. B, Hormone induction of the LH surge caused a significant increase in the percentage of pubertally born BrdU-ir cells that express Fos-ir (*p ≤ 0.0001). The graph represents mean ± SEM; for oil treated, n = 6, for hormone treated, n = 7.
- Figure 2.
Some pubertally born cells were ERα-ir, but none were PR-ir. Z-stack images in the AVPV of BrdU-ir cells colocalized with nuclear (top panel) and membrane-associated ERα-ir (middle panel). BrdU did not colocalize with PR (bottom panel). BrdU, ERα, mERα, PR, and merge images are maximum intensity projections of 30 images (15 μm thick, 0.5-μm step). White scale bars, 10 μm. In the column on the right, orthogonal views through the middle of the cells confirm colocalization (top two panels) or lack of colocalization (bottom panel); red arrow, x plane; green arrow, y plane; and blue arrow, z plane. Fluorescent labeling for BrdU/ERα performed on tissue from four ovariectomized and vehicle-treated rats; labeling for BrdU/PR was performed on tissue from four ovariectomized and estradiol- and P-treated rats.
- Figure 3.
Fewer cells were added to the AVPV during adulthood than during puberty and the mitotic inhibitor, AraC, reduced the number of BrdU-ir cells in the AVPV. A, Representative images show BrdU-ir cells in the AVPV of female rats treated with vehicle (BrdU in aCSF) or AraC (in BrdU and aCSF vehicle) during puberty or adulthood. Scale bar, 100 μm. B, Graph depicts the quantitative results showing that on average, more cells were added to the AVPV during puberty than adulthood (main effect of age during treatment, *p = 0.017) and that on average, AraC reduced the number of BrdU-ir cells added to the AVPV (main effect of treatment, **p = 0.001). Graph represents mean ± SEM of the total number per animal of BrdU-ir cells in four anatomically matched sections through the AVPV; n = 7 for pubertal control, n = 5 for pubertal AraC, n = 6 for adult control, n = 7 for adult AraC.
- Figure 4.
Both pubertally born and adult born cells in the AVPV were activated in adulthood by hormone treatments designed to induce an LH surge. The graph depicts the percentage of pubertally or adult-born (BrdU-ir) cells in the AVPV that also express Fos-ir during the time of the LH surge in adult female rats that were treated with BrdU during puberty or during adulthood. Data are presented as mean ± SEM; n = 3/age.
- Figure 5.
Some cells in the AVPV that were born during puberty or adulthood had differentiated by three weeks, with or without AraC-treatment. A, Representative photomicrographs of pubertally born cells that were NeuN-ir or GFAP-ir. Images are maximum intensity projections of a Z-stack (0.5-μm step). Scale bars, 10 μm. Orthogonal views through the middle of the cells (right column) confirm colocalization; red arrow, x plane; green arrow, y plane; and blue arrow, z plane. B, Histograms represent the mean proportion of BrdU-ir cells that express GFAP, NeuN, or only BrdU-immunoreactivity in animals receiving either vehicle (BrdU in aCSF) or AraC (AraC + BrdU in aCSF) ICV during puberty or adulthood (n = 2/age/treatment).
- Figure 6.
Knockdown of cell addition with AraC during puberty or adulthood reduced and delayed the LH surge. A, Experimental timeline. Pubertal (4 week old) or adult (9-10 week old) female rats received ICV cannulae with osmotic minipumps containing AraC (33 μg/μl) and BrdU (4.5 μg/μl) in aCSF (AraC treatment group), or just BrdU in aCSF (control treatment group). Females were ovariectomized (OVX) after the end of ICV infusion and then primed with EB (10 μg in 0.05 ml sesame oil; s.c.) for two consecutive days at 10 A.M., and then P (500 μg in 0.1 ml sesame oil; s.c.) on the third day at 10 A.M. Blood sampling occurred on the same day as P injection, from 11 A.M. to 7 P.M. Pubertal (B) or adult (C) AraC treatment reduced the LH surge across time (p = 0.047). Dotted vertical lines indicate lights off. Symbols above points indicate significant differences observed with Fishers LSD post hoc comparisons. D, AraC treatment reduced the maximum LH concentration (p = 0.022) compared with that observed in control animals. E, The LH peak was delayed in AraC-treated animals (p = 0.05), irrespective of age during treatment. Graphs represent mean ± SEM; n = 4/group, except for pubertal-AraC (n = 3); *p ≤ 0.05, +p ≤ 0.01.
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