Figures and data in CRISPR-Cas9 knockdown of ESR1 in preoptic GABA-kisspeptin neurons suppresses the preovulatory surge and estrous cycles in female mice

Figures
Tables
Additional files

5 figures, 2 tables and 2 additional files

Figures

Figure 1

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CRISPR knockdown of ESR1 in preoptic GABA neurons.

(**A, B**) Photomicrographs showing distribution of mCherry (gRNA from AAV) and expression of GFP (Cas9) in VGAT neurons and nuclear-located ESR1 (blue) in the preoptic periventricular (PVpo) of two mice receiving either gRNA-LacZ (A) or gRNA-2 (B). The mCherry signal is removed in the adjoining plates (a) and (b) so that the VGAT neurons (green) co-expressing ESR1 (blue or teal nuclei) are more easily identified. Scale bar in (a) is the same for all photomicrographs. (C) Individual data points (n=6-9) and mean ± SEM percentage of GFP/VGAT neurons expressing ESR1 within injected regions of the RP3V and medial preoptic nuclei (MPN) for the three gRNA groups. ***p<0.001 versus gRNA-LacZ (ANOVA, post hoc Dunnett’s tests). Data in Source data 1. (**D–G**) Representative schematics of AAV injection sites (pink) in four mice; 16,337 (bilateral gRNA-2), 14,945 (unilateral RP3V, bilateral MPN gRNA-2), 13,631 (bilateral gRNA-3), 13,786 (bilateral gRNA-LacZ).

Figure 2

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Deletion of ESR1 from preoptic GABA neurons and estrous cyclicity.

(**A–C**) Individual paired data points (n=6-7) and mean ± SEM estrous cycle length before and after AAV gRNA injection of lacZ, gRNA-2, and gRNA-3 into the RP3V and medial preoptic nuclei (MPN). (**D–E**) Individual paired data points (n=9-10) and mean ± SEM estrous cycle length before and after AAV gRNA injection in mice with bilateral AAV injections in the RP3V and MPN analyzed separately. Four mice (3× gRNA-2, 1× gRNA-3) enter constant estrus and one gRNA-2 mouse was in constant diestrus, all scored as a cycle length of 0. No significant effects of gRNA injection were detected (p>0.05 Wilcoxon paired tests). (**F–H**), Examples of estrous cycle patterns from three mice including one (G) that entered constant estrous following gRNA-2 injection. The individual animal number is given in each frame. All data in Source data 1.

Figure 3

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Deletion of ESR1 from preoptic GABA neurons does not alter pulsatile luteinizing hormone (LH) secretion.

(**A–D**) Representative LH pulse profiles from female mice given AAV gRNA-lacZ, gRNA-2, and gRNA-3. The mouse identification number is given in brackets. (**E–G**) Histograms show the individual data points (n=5-6) and mean ± SEM for parameters of pulsatile LH secretion in mice given gRNA-lacZ, gRNA-2, and gRNA-3 into the RP3V and medial preoptic nuclei (MPN). No significant effects are detected (p>0.05, Kruskal-Wallis test). (**H–J**) Correlations between the % VGAT neurons with ESR1 in the RP3V and parameters of pulsatile LH secretion. Individual mice are color-coded according to their gRNA treatment. No significant correlations were detected (Pearson r<0.34 in all cases). (**K–M**) Correlations between the % VGAT neurons with ESR1 in the MPN and parameters of pulsatile LH secretion. Individual mice are color-coded according to their gRNA treatment. No significant correlations were detected (Pearson r<0.41 in all cases). All data in Source data 1.

Figure 4

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Effects of ESR1 deletion in preoptic GABA neurons on surge parameters.

(**A, B**) Individual data points (n=6-7) and mean ± SEM values showing the percentage of gonadotropin-releasing hormone (GnRH) neurons with cFos and single-point luteinizing hormone (LH) levels for mice killed at the time of the expected surge given gRNA-LacZ (black), gRNA-2 (blue), and gRNA-3 (green) injections centered on the RP3V and medial preoptic nuclei (MPN). **p=0.0023 (Krusakl-Wallis) compared with LacZ. (**C, D**) Correlations between the % of RP3V VGAT neurons with ESR1 and cFos expression by GnRH neurons or LH secretion. Individual mice are color-coded according to their gRNA treatment. A significant correlation for cFos in GnRH neurons exists (p=0.008, Pearson r=0.66) but not for LH (p=0.26, Pearson r=0.31). (**E, F**) Correlations between the % of MPN VGAT neurons with ESR1 and cFos expression by GnRH neurons or LH secretion. Individual mice are color-coded according to their gRNA treatment. No significant correlations were found. All data in Source data 1.

Figure 5

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Suppression of RP3V kisspeptin expression is associated with the loss of estrous cycles and the surge mechanism.

(**A, B**) Dual-label immunohistochemistry for kisspeptin (brown) and ESR1 (black) shows the normal high level of co-expression (white arrowheads) in a representative gRNA-lacZ mouse (A) but near absence of kisspeptin immunoreactivity in a representative ‘bilateral loss’ mouse (#16337) (B). 3V, third ventricle. Scale bar in B is the same for A. (C) Individual data points (n=4-7) and mean ± SEM values showing estrous cycle length before and after gRNA injection in gRNA ‘lacZ’ mice, gRNA-2/3 mice with ‘normal’ kisspeptin expression, gRNA-2/3 mice with a ‘unilateral’ reduction in kisspeptin, and gRNA-2/3 mice with a near-complete ‘bilateral’ loss of kisspeptin. *p<0.05 compared to pre-values. (**D, E**) Individual data points (n=4-7) and mean ± SEM single-point luteinizing hormone (LH) levels and % of gonadotropin-releasing hormone (GnRH) neurons with cFos at the time of the expected surge in gRNA ‘lacZ’ mice, gRNA-2/3 mice with ‘normal’ kisspeptin expression, gRNA-2/3 mice with a ‘unilateral’ reduction in kisspeptin, and gRNA-2/3 mice with a near-complete ‘bilateral’ loss of kisspeptin. *p<0.05 compared to lacZ. (**F–H**) Individual data points (n=3-7) and mean ± SEM parameters of pulsatile LH secretion in gRNA ‘lacZ’ mice, gRNA-2/3 mice with ‘normal’ kisspeptin expression, gRNA-2/3 mice with a ‘unilateral’ reduction in kisspeptin, and gRNA-2/3 mice with a near-complete ‘bilateral’ loss of kisspeptin. No significant differences were detected. All data in Source data 1.

Tables

Table 1

Kisspeptin-ESR1 co-expression in re-grouped gRNA mice.

Table showing the numbers of kisspeptin neurons/section in the anteroventral periventricular (AVPV) and preoptic periventricular (PVpo) and percentage expression with ESR1. ‘LacZ’ refers to all mice given gRNA-LacZ, ‘normal’ refers to all gRNA-2/3 mice with normal kisspeptin expression (unilateral cell counts shown), ‘unilateral’ refers to gRNA-2/3 mice in which only one side of the brain had reduced kisspeptin cell numbers with the cell count on the affected side given, ‘bilateral’ refers to gRNA-2/3 mice with essentially no cytoplasmic kisspeptin expression bilaterally in the RP3V. Too few kisspeptin neurons existed to reliably determine co-expression with ESR1. **p<0.01, ***p<0.001 compared with lacZ group (Kruskal-Wallis with Dunn’s tests, exact p-values given below). All data in Source data 1.

		LacZ (n=8)	Normal (n=7)	Unilateral(n=6)	Bilateral (n=4)
AVPV	No. kisspeptin neurons/section	20.1±2.0	15.4±0.9	5.6±2.5** (p=0.0066)	0.4±0.1*** (p=0.0006)
AVPV	% Kiss with ESR1	74.5±3.3	78.5±4.4	57.3±19.7	–
PVpo	No. kisspeptin neurons/section	24.0±1.9	19.6±1.3	4.8±2.8** (p=0.0016)	0.8±0.3** (p=0.0031)
PVpo	% Kiss with ESR1	63.3±5.1	70.5±5.2	27.5±17.1	–

Table 2

VGAT-ESR1 in mice grouped on the basis of kisspeptin expression.

Table showing the percentage of VGAT neurons in the RP3V and MPN expressing ESR1 in gRNA mice re-grouped on the basis of kisspeptin expression (see Table 1 for explanation of groups). *p<0.05, ***p<0.001 compared with lacZ group (Kruskal-Wallis with Dunn’s tests, exact p-values given below). All data in Source data 1.

		LacZ (n=8)	Normal (n=7)	Unilateral (n=6)	Bilateral (n=4)
RP3V	% VGAT with ESR1	50.4±6.1	17.9±2.3* (p=0.0242)	17.7±3.0* (p=0.0358)	24.5±4.8
MPN	% VGAT with ESR1	61.2±4.2	14.0±3.1*** (p=0.0002)	22.0±5.1* (p=0.0162)	30.1±4.8