Female HPA axis displays heightened sensitivity to pre-pubertal stress

Figures & data

Table 1. PCR primers.

Target	Sequence
GR	F: 5′-AGCACAATTACCTTTGTGCTGGA R: 5′- TTCGATAGCGGCATGCTGGA
MR	F: 5′-AATAACGTCCCTCTGCGCTC R: 5′- GCCTGAAGTGGCATAGCTGA
FKBP5	F: 5′-GAACCCAATGCTGAGCTTA R: 5′-ATGTACTTGCCTCCCTTGAA
AVP	F: 5′-GCTGCTGCTTAGGCTGGTACA R: 5′-AGTTTATTTTCCATGCTGTAGGGGG
OXT	F: 5′-CTGCTGTAGCCCGGATGG R: 5′-AAGGAAGCGCCCTAAAGGTA
AVPR1a	F: 5′-AGCGCCTACATCCTTTGCTG R: 5′-TGGAAGGGTTTTCTGAATCGGTC
OXTR	F: 5′-CAAGGAAGCTTCTGCCTTCATC A R: 5′-GACGGCTCAGGCACAAAGGTG
Hprt1	F: 5′-TCCTCCTCAGACCGCTTTTC R: 5′-ATCACTAATCACGACGCTGGG
Gapdh	F: 5′-TCTCTGCTCCTCCCTGTTCT R: 5′-TACGGCCAAATCCGTTCACA

F: forward; R: reverse primer.

Table 2. Full statistical report.

Area	Factor	Group	Sex	Time of sacrifice	Group*sex	Group*time of sacrifice	Sex*time of sacrifice	Group*sex*time of sacrifice
Hippocampal formation
GR		F1,7.63 = 0.01, p = .92	F1,25.66 = 1.18, p = .29	N/A	F1,25.66 = 0.24, p = .63	N/A	N/A	N/A
MR		F1,9.44 = 0.86, p = .38	F1,29.54 = 0.5, p = .48	N/A	F1,29.54 = 1.88, p = .18	N/A	N/A	N/A
GR:MR		F1,8.06 = 0.88, p = .38	F1,27.4 = 0.64, p = .43	N/A	F1,27.4 = 0.45, p = .51	N/A	N/A	N/A
AVP		F1,10.93 = 0.62, p = .45	F1,30.02 = 1.2, p = .28	N/A	F1,30.02 = 0.57, p = .45	N/A	N/A	N/A
AVPR1a		F1,10.66 = 2.41, p = .15	F1,26.64 = 2.1, p = .16	N/A	F1,26.64 = 4.47, p = .04	N/A	N/A	N/A
OXT		F1,9.56 = 0.26, p = .62	F1,28.54 = 1.92, p = .18	N/A	F1,28.54 = 0.9, p = .35	N/A	N/A	N/A
OXTR		F1,9.84 = 0.02, p = .9	F1,25.67 = 0.02, p = .89	N/A	F1,25.67 = 0.01, p = .93	N/A	N/A	N/A
FKBP5		F1,4.22 = 5.79, p = .07	F1,29.85 = 1.81, p = .19	N/A	F1,29.85 = 4.33, p = .04	N/A	N/A	N/A
PFC
GR		F1,5.78 = 3, p = .14	F1,24.94 = 2.32, p = .14	N/A	F1,24.94 = 7.17, p = .01	N/A	N/A	N/A
MR		F1,8.81 = 0.14, p = .72	F1,24.86 = 1.35, p = .26	N/A	F1,24.86 = 3.69, p = .07	N/A	N/A	N/A
GR:MR		F1,9.17 = 2.4, p = .15	F1,25.37 = 9.66, p = .005	N/A	F1,25.37 = 4.97, p = .03	N/A	N/A	N/A
AVP		F1,9.77 = 0.87, p = .37	F1,29.41 = 0.03, p = .87	N/A	F1,29.41 = 0.14, p = .71	N/A	N/A	N/A
AVPR1a		F1,8.62 = 0.74, p = .41	F1,23.17 = 0.17, p = .68	N/A	F1,23.17 = 6.94, p = .01	N/A	N/A	N/A
OXT		F1,10.34 = 0.17, p = .68	F1,27.16 = 2.11, p = .16	N/A	F1,27.16 = 0.13, p = .72	N/A	N/A	N/A
OXTR		F1,10.39 = 0.16, p = .7	F1,27.9 = 28.65, p < .0001	N/A	F1,27.9 = 0.01, p = .94	N/A	N/A	N/A
FKBP5		F1,9.23 = 1.43, p = .26	F1,25.6 = 3.29, p = .08	N/A	F1,25.6 = 1.72, p = .2	N/A	N/A	N/A
Hypothalamus
GR		F1,10.79 = 0.08, p = .78	F1,30.7 = 13.68, p < .001	N/A	F1,30.7 = 0.05, p = .83	N/A	N/A	N/A
MR		F1,9 = 0.53, p = .49	F1,25.52 = 63.73, p < .0001	N/A	F1,25.52 = 2.21, p = .15	N/A	N/A	N/A
GR:MR		F1,11.03 = 0.15, p = .71	F1,28.33 = 8.93, p < .01	N/A	F1,28.33 = 2.95, p = .1	N/A	N/A	N/A
AVP		F1,7.69 = 0.86, p = .38	F1,31.66 = 0.005, p = .94	N/A	F1,31.66 = 0.44, p = .51	N/A	N/A	N/A
AVPR1a		F1,10.42 = 0.04, p = .84	F1,25.55 = 27.06, p < .0001	N/A	F1,25.55 = 0.01, p = .91	N/A	N/A	N/A
OXT		F1,10.33 = 0.4, p = .54	F1,26.76 = 42.06, p < .0001	N/A	F1,26.76 = 0.01, p = .91	N/A	N/A	N/A
OXTR		F1,10.84 = 0.43, p = .53	F1,29.24 = 118.81, p < .0001	N/A	F1,29.24 = 1.2, p = .28	N/A	N/A	N/A
FKPB5		F1,10.64 = 0.63, p = .44	F1,26.23 = 43.7, p < .0001	N/A	F1,26.23 = 0.26, p = .61	N/A	N/A	N/A
Corticosterone		F1,14.15 = 2.57, p = .13	F1,53.98 = 1.22, p = .27	F1,47.29 = 22.51, p < .0001	F1,53.98 = 0.88, p = .35	F1,47.29 = 0.83, p = .37	F1,53.32 = 2.39, p = .13	F7,37.77 = 3.53, p = .0052

Significant terms are shown in bold.

Figure 1. Hippocampal formation. PPS increased expression of (a) FKBP5 and (b) AVPR1a in the female hippocampal formation. Con: control; PPS: pre-pubertal stress; F: female; M: male. Male: 12 control; 10 PPS; female: 8 control; 10 PPS. *p < .05. Error bars represent 1 S.E. and bars joined by a line and asterisk are significantly different to one another.

Figure 2. PFC. PPS increased (a) GR and (b) GR:MR ratio and decreased (c) AVPR1a in the female PFC. OXTR expression was higher in female than male PFC. Con: control; PPS: pre-pubertal stress; F: female; M: male. Male: 12 control; 10 PPS; female: 8 control; 10 PPS. *p < .05, **p < .01, ***p < .0001. Error bars represent 1 S.E. and bars joined by a line and asterisk are significantly different to one another.

Figure 3. Hypothalamus. (a) GR, (b) MR, (c) GR:MR, (d) FKBP5 and (e) OXTR were higher in male than female hypothalamus, whereas (f) AVPR1a and (g) OXT were higher in female hypothalamus. Con: control; PPS: pre-pubertal stress; F:, female; M: male. Male: 12 control; 10 PPS; female: 8 control; 10 PPS. **p < .01, ***p < .0001. Error bars represent 1 S.E. and bars joined by a line and asterisk are significantly different to one another.

Table 3. Summary of gene expression and CORT changes as a result of PPS and sex differences.

Region	Target	PPS vs. Con		Female vs. male
		Female	Male
Hippocampus	AVP	/	/	/
	AVPR1a	+	/	/
	OXT	/	/	/
	OXTR	/	/	/
	GR	/	/	/
	MR	/	/	/
	GR:MR	/	/	/
	FKBP5	+	/	/
PFC	AVP	/	/	/
	AVPR1a	−	/	/
	OXT	/	/	/
	OXTR	/	/	+
	GR	+	/	/
	MR	/	/	/
	GR:MR	+	/	/
	FKBP5	/	/	/
Hypothalamus	AVP	/	/	/
	AVPR1a	/	/	+
	OXT	/	/	+
	OXTR	/	/	−
	GR	/	/	−
	MR	/	/	−
	GR:MR	/	/	−
	FKBP5	/	/	−
Basal	Corticosterone	/	/	/
Social	Corticosterone	−	/	/

+ = increased expression, – = decreased expression, and /= no change in expression in PPS compared to controls or in females compared to males.

Figure 4. Corticosterone. Social interaction significantly elevated corticosterone above baseline in control animals and PPS males. This response was blunted in PPS females. Con: control; PPS: pre-pubertal stress; F: female; M: male. Females: 22 control; 18 PPS; male: 12 control; 10 PPS *p < .05. Error bars represent 1 S.E. and bars joined by a line and asterisk are significantly different to one another.