Prepubertal Development of Gonadotropin-Releasing Hormone Neuron Activity Is Altered by Sex, Age, and Prenatal Androgen Exposure

Abstract

Gonadotropin-releasing hormone (GnRH) neurons regulate reproduction though pulsatile hormone release. Disruption of GnRH release as measured via luteinizing hormone (LH) pulses occurs in polycystic ovary syndrome (PCOS), and in young hyperandrogenemic girls. In adult prenatally androgenized (PNA) mice, which exhibit many aspects of PCOS, increased LH is associated with increased GnRH neuron action potential firing. How GnRH neuron activity develops over the prepubertal period and whether this is altered by sex or prenatal androgen treatment are unknown. We hypothesized GnRH neurons are active before puberty and that this activity is sexually differentiated and altered by PNA. Dams were injected with dihydrotestosterone (DHT) on days 16 to 18 post copulation to generate PNA mice. Action potential firing of GFP-identified GnRH neurons in brain slices from 1-, 2-, 3-, and 4-week-old and adult mice was monitored. GnRH neurons were active at all ages tested. In control females, activity increased with age through 3 weeks, then decreased to adult levels. In contrast, activity did not change in PNA females and was reduced at 3 weeks. Activity was higher in control females than males from 2 to 3 weeks. PNA did not affect GnRH neuron firing rate in males at any age. Short-term action potential patterns were also affected by age and PNA treatment. GnRH neurons are thus typically more active during the prepubertal period than adulthood, and PNA reduces prepubertal activity in females. Prepubertal activity may play a role in establishing sexually differentiated neuronal networks upstream of GnRH neurons; androgen-induced changes during this time may contribute to the adult PNA, and possibly PCOS, phenotype.

Figure 1.

Characterization of PNA animals. (A) Age and (B) body mass at vaginal opening in control (open symbols) and PNA (filled symbols) females. (C) Age and (D) body mass at preputial separation in control and PNA males. (E) Anogenital distance in adult female littermates of mice that were used for recording before puberty. (F) Representative estrous cycles in adult female littermates: *P < 0.05 control vs PNA, unpaired Student t test in all but (A) in which a Mann-Whitney U test was used. D, diestrus; E, estrus; P, proestrus.

Figure 2.

Activity of GnRH neurons from female mice changes with age and prenatal androgenization. One-minute representative raw recordings of GnRH neurons from control (left) and PNA (right) females.

Figure 3.

GnRH neuron activity changes throughout the prepubertal period in both sexes and is altered by PNA. (A–D) Individual values and mean ± SEM of firing rate at 1, 2, 3, and 4 weeks of age and adults. (A) Control females (open circles) and males (black circles); (B) control and PNA (magenta circles) females; (C) control and PNA (blue circles) males; (D) PNA females and control males. Different letters of the same case indicate differences with age within a group; there were no changes with age in female PNA mice (P ≥ 0.0978). *P < 0.05, ^#P < 0.01 between treatment groups at each age. Two-way ANOVA/Fisher LSD for comparisons among all groups within sex; two-way ANOVA/Sidak for comparisons at the same age between sexes (control female vs control male; PNA female vs control male). CON, control.

Figure 4.

Spike timing in GnRH neurons changes with age in females and differs with PNA treatment in adults. (A) Log₁₀ ISI distributions of GnRH recordings for each group of female mice. (B) Log₁₀ ISI distribution for each group of male mice. Arrow indicates longer interval shoulder (see text). (C) Log₁₀ ISI distributions comparing male control and female PNA mice, *P < 0.05, KS. Note change in color in panel (C) vs (A) and (B) for adult animals. CON, control.

Figure 5.

Burst patterns of GnRH neurons differ with age but are not affected by PNA. (A) Schematic showing burst parameter determination. (B) Burst frequency, (C) duration, (D) and spikes/burst in cells from female mice. (E) Burst frequency, (F) duration, and (G) spikes/burst in cells from male mice. *P < 0.05, two-way-ANOVA/Tukey.