Article Figures & Data
Figures
- Figure 1.
Brief direct infusion of EB into the S-ME induces GnRH release. A–D, Representative cases (A, B) and group data (means ± SEM) from EB-infused (C) or vehicle-infused (D) animals are shown. Time 0 designates the beginning of a 20 min EB or vehicle infusion (gray bars). Two-way ANOVA indicates that EB significantly stimulated GnRH release (C) over vehicle control (D, p = 0.0018). Post hoc analysis further indicates that GnRH levels during the first 10 min of EB infusion were significantly higher than those during the control period (C; **p < 0.01) as well as the corresponding time period of vehicle infusion (D; #p < 0.05). Arrowheads indicate pulses identified by Pulsar.
- Figure 2.
ES, but not sham ES, stimulates GnRH release (black line with filled circle), and E2 release after an initial suppression (red line with filled square). A–E, A representative case (A) and group data (means ± SEM) from ES (B, D) or sham ES (C, E) are shown. Time 0 designates the beginning of a 5 min ES or sham ES treatment (gray bars). Note that there were GnRH pulses and small E2 fluctuations before ES application and that ES stimulated GnRH release, which was followed by E2 release. Sham ES did not induce any significant change. Two-way ANOVA analysis further indicated that ES significantly stimulated release of GnRH (B; p = 0.0006) and E2 (D; p = 0.047) over sham controls (C, E). Post hoc test indicated that GnRH increases at 0–20 and 20–40 min after the initiation of ES were significantly higher (***p < 0.001 and *p < 0.05, respectively; B) than before ES application and also significantly higher (###p < 0.001 and #p < 0.05; B vs C) than those in sham ES at the corresponding time period. Similarly, the E2 increase at 40–60 min after ES was significantly higher (**p < 0.01) than before ES (D) and also significantly higher (##p < 0.01; D vs E) than sham ES at the corresponding time period. Interestingly, the E2 values at 0–20 and 20–40 min after ES were significantly lower than during 40–60 min (aaap < 0.001 and aaap < 0.001, respectively), but neither value was significantly lower than before ES (p > 0.05 and p > 0.05, respectively). Arrowheads indicate pulses identified by Pulsar.
- Figure 3.
Direct infusion of EB into the S-ME induces release of GnRH (black line with filled circle) and E2 (red line with filled square) in vivo. A–F, Representative cases (A, B) and group data (means ± SEM) from EB-infused (C, E) or vehicle-infused (D, F) animals are shown. Time 0 designates the beginning of a 20 min EB (10 or 100 nm) or vehicle infusion (gray bars). Note that EB induced increases in GnRH release and pulsatile E2 release (A). While the amplitude of GnRH responses appears to be EB dose-dependent, the amplitude of E2 did not vary. In contrast, vehicle infusion did not cause any significant effects (B). In this case, a single spontaneous E2 peak was seen at 8 h. Two-way ANOVA analysis further indicated that EB (100 nm) significantly stimulated GnRH release (C; p = 0.0098) as well as E2 release (E; p = 0.0009) over respective vehicle controls. Post hoc test further indicated that the GnRH increases at 0–20 and 20–40 min after EB infusion were significantly higher (***p < 0.001 and *p < 0.05, respectively) than before EB infusion (C) and the GnRH increase at 0–20 min after EB infusion was also significantly higher (##p < 0.01) than after vehicle infusion at the corresponding time period (C vs D). Similarly, the E2 increase at 0–20 min after EB infusion was significantly higher (***p < 0.001) than before EB infusion (E) and also significantly higher (###p < 0.001) than after vehicle infusion at the corresponding period (E vs F). Arrowheads indicate pulses identified by Pulsar.
- Figure 4.
Infusion of letrozole (100 nm, light gray bars) into the S-ME inhibits spontaneous GnRH pulses as well as the EB-induced GnRH and E2 increases. A–G, An example (A) and the results of two-way ANOVA examining the effects of EB (B, E), of letrozole on EB-induced release (C, F), and of letrozole on spontaneous release (D, G) are shown. In A, an EB challenge (100 nm, dark gray bars) was first given for 20 min with vehicle. Subsequently letrozole (100 nm, light gray bars) was continuously infused for 100 min, during which EB (100 nm) infusion was added for 20 min (60–80 min after the initiation of letrozole infusion), and finally letrozole (100 nm) alone was infused for 80 min. Note that control EB infusion stimulated release of GnRH and E2, whereas EB infusion in the presence of letrozole failed to stimulate release of GnRH or E2 (A). Likewise, letrozole infusion alone blocked spontaneous GnRH pulses (A). ANOVA analysis indicated that letrozole in the presence of EB (C) or letrozole alone (D) significantly reduced mean GnRH release compared with EB control (B; p < 0.0001). Similarly, letrozole in the presence of EB (F) or letrozole alone (G) significantly reduced mean E2 release compared with EB control (E; p < 0.0001). Post hoc test indicated that GnRH levels during control EB infusion (B) and during the letrozole infusion with (C) or without (D) EB were all significantly different from respective control (before) periods (B–D; **p < 0.01 for EB alone, **p < 0.01 for letrozole plus EB, *p < 0.05 for letrozole alone). GnRH levels during both letrozole treatments were significantly lower than those during EB with vehicle (aaap < 0.001, ap < 0.05 for letrozole plus EB, aaap < 0.001 for letrozole alone). Likewise, E2 levels during both letrozole treatments (F, G) were lower than those during EB control (E; aaap < 0.001 for letrozole plus EB; aaap < 0.001 for letrozole alone). E2 levels during EB with vehicle treatment (E) were also significantly higher than before infusions (**p < 0.01).
- Figure 5.
A–G, Midsagittal diagram of the hypothalamus showing locations of the microdialysis probe tip (A) and comparison of probe tip position with GnRH (B, D, F) and E2 levels (C, E, G). The distance from the midline (B, C), the anterior–posterior position (D, E), and the dorsal–ventral position (F, G) in reference to the infundibular recess of the third ventricle are shown. The vertical and horizontal lines in A indicate the dorsal–ventral and anterior–posterior ranges of the sampling locations. The intersections of these lines indicate the mean cannula location, and the box delineates the SEM. The lateral position (from midline; mean ± SEM) was 0.5 ± 0.1 mm. GnRH levels 0–1 mm posterior were significantly >0–1 mm anterior from the infundibular recess (p = 0.0471 with unpaired Student's t test) and GnRH levels 0–1 mm ventral were significantly >0–1 mm dorsal from the infundibular recess (p = 0.0241 with unpaired Student's t test). E2 levels at 0.5–1 mm were significantly higher than at 0–0.5 mm lateral from midline (p = 0.0461 with unpaired Student's t test). The number of experiments in this mapping was 31. ap, Anterior pituitary; ir, infundibular recess; mm, mammillary body; oc, optic chiasm; pp, posterior pituitary; V III, third ventricle.
Tables
E2 recovery through probe membrane (number of experiments) Peak recovery rate (%) Anterodialysis/E2 uptake from reservoir (n = 3) 13.13 ± 2.39 Retrodialysis/E2 infusion (n = 3) 10.81 ± 0.52 ↵aThe recovery rate of E2 was assessed in vitro by (1) infusing artificial CSF through microdialysis probes placed in a reservoir containing E2 at 100 nm while dialysates were continuously collected at 10 min intervals or (2) infusing E2 at 100 nm for 20 min through microdialysis probes into a reservoir containing artificial CSF, with the reservoir changed every 10 min.
- Table 2.
Effects of ES and EB infusion of the MBH-ME region on release of GnRH and E2 in OVX female rhesus monkeys
Measured neurohormones GnRH E2 Treatments ES (N = 9) Sham ES (N = 9) ES (N = 9) Sham ES (N = 9) ES Peak before (pg/ml) 3.1 ± 0.4 2.9 ± 1.0 242 ± 63 (5)* 236 ± 37 (4)* Peak after (pg/ml) 14.0 ± 2.7aaa,bbb 3.3 ± 0.9 437 ± 20a,b 232 ± 72 (4)* Peak latency (min) 24.4 ± 2.9a,ccc 52.5 ± 10.0 64.4 ± 4.4 44.0 ± 8.4 (4)* Peak duration (min) 42.2 ± 5.2 44.4 ± 7.3 37.8 ± 5.2 40.0 ± 8.2 (4)* IPI (min) 66.7 ± 4.7 62.9 ± 8.1 64.0 ± 7.5 (5)* 60.0 ± 8.2 (4)* Treatments EB (N = 10) Vehicle (N = 10) EB (N = 10) Vehicle (N = 10) EB Peak before (pg/ml) 3.1 ± 0.4 3.7 ± 0.4 281 ± 129 (5)* 265 ± 80 (5)* Peak after (pg/ml) 14.8 ± 4.3aa,bb 3.3 ± 0.6 1285 ± 285a,b 281 ± 53 (5)* Peak latency (min) 26.0 ± 3.1aa 58.0 ± 9.6 25.5 ± 2.8aa 48.0 ± 4.9 (5)* Peak duration (min) 38.0 ± 5.5 44.0 ± 5.8 32.0 ± 4.4 32.0 ± 3.3 (5)* IPI (min) 62.0 ± 6.0 66.0 ± 5.0 70.0 ± 16.5 (8)* 65.0 ± 12.6 (5)* ↵Data are mean ± SEM, N: Total number of experiments conducted. ()*: Number of experiments used for calculation; in some experiments sufficient E2 peaks were not present for calculation.
↵a p < 0.05;
↵aa p < 0.01;
↵aaa p < 0.001 versus vehicle or sham;
↵b p < 0.05;
↵bb p < 0.10;
↵bbb p < 0.001 versus mean before treatment;
↵ccc p < 0.001 versus E2.
- Table 3.
The effects of the interval between experiments and the total number of experiments on E2 and GnRH levels
Experimental conditions Mean E2 (pg/ml) Mean GnRH (pg/ml) Intervals between two probe insertions 0–21 d from last experiment (n = 17) 127 ± 58a 4.48 ± 1.46b 22–50 d from last experiment (n = 14) 152 ± 59a 3.26 ± 0.41b Total number of experiments 1–5 experiments conducted (n = 9) 118 ± 58c 3.75 ± 1.28d 5–10 experiments conducted (n = 8) 179 ± 136c 2.24 ± 0.36d 10–15 experiments conducted (n = 14) 139 ± 59c 4.54 ± 1.38d Retrospective analyses indicate that, reactive glia are not a likely a source of E2 release in the S-ME, as neither time between two microdialysis experiments nor the number of experiments were correlated with mean E2 and GnRH levels. The same letter indicates no difference between conditions by Student's t test (for the intervals between two probe insertions) or 1-way ANOVA (for the total number of experiments).
↵a: Mean E2 (p = 0.7658);
↵b: Mean GnRH (p = 0.4575);
↵c: Mean E2 (p = 0.8874);
↵d: Mean GnRH (p = 0.5967). Note that the total number of experiments can reach up to 15 times per animal, as the same animals were also co-assigned to two additional unrelated microdialysis sampling projects.
Conditions tested for EB to E2 conversion Total number of cases examined Number of cases with E2 peak present Peak E2 levels (pg/ml) Conversion rate (%) Artificial CSF 3 0 na 0 Monkey plasma 3 0 na 0 Monkey CSF 4 2 75, 90 0.28, 0.52 Hypothalamic tissues 3 1 2 0.02 Because E2 increases induced by EB infusion could be due to hydrolysis and/or conversion of EB by an endogenous esterase, we conducted a series of validation experiments. We infused EB at 100 nm for 20 min (2 μl/min speed) through a microdialysis probe in 1) artificial CSF, 2) monkey plasma, or 3) monkey CSF obtained by spinal tap, while dialysates were continuously collected.
