Prenatal androgenization of female mice programs an increase in firing activity of gonadotropin-releasing hormone (GnRH) neurons that is reversed by metformin treatment in adulthood

Abstract

Prenatal androgenization (PNA) of female mice with dihydrotestosterone programs reproductive dysfunction in adulthood, characterized by elevated luteinizing hormone levels, irregular estrous cycles, and central abnormalities. Here, we evaluated activity of GnRH neurons from PNA mice and the effects of in vivo treatment with metformin, an activator of AMP-activated protein kinase (AMPK) that is commonly used to treat the fertility disorder polycystic ovary syndrome. Estrous cycles were monitored in PNA and control mice before and after metformin administration. Before metformin, cycles were longer in PNA mice and percent time in estrus lower; metformin normalized cycles in PNA mice. Extracellular recordings were used to monitor GnRH neuron firing activity in brain slices from diestrous mice. Firing rate was higher and quiescence lower in GnRH neurons from PNA mice, demonstrating increased GnRH neuron activity. Metformin treatment of PNA mice restored firing activity and LH to control levels. To assess whether AMPK activation contributed to the metformin-induced reduction in GnRH neuron activity, the AMPK antagonist compound C was acutely applied to cells. Compound C stimulated cells from metformin-treated, but not untreated, mice, suggesting that AMPK was activated in GnRH neurons, or afferent neurons, in the former group. GnRH neurons from metformin-treated mice also showed a reduced inhibitory response to low glucose. These studies indicate that PNA causes enhanced firing activity of GnRH neurons and elevated LH that are reversible by metformin, raising the possibility that central AMPK activation by metformin may play a role in its restoration of reproductive cycles in polycystic ovary syndrome.

Fig. 1.

Timeline of metformin administration and physiological assessments. cycles, Estrous cycle monitoring.

Fig. 2.

Metformin has minimal effects on metabolic parameters in PNA mice. A, Average daily food intake per unit body mass was similar in PNA and CON mice and unaffected by metformin (met) (n = 11–12 per group). Arrow indicates start of metformin administration to treated groups. B, Body mass was unaffected by metformin (n = 11–12 per group). Arrow indicates start of treatment. C, Ratio of parametrial fat pad mass to body mass was not different among groups (n = 21–24 per group). D, Blood glucose levels in random-fed (n = 21–24 per group) and fasted (n = 10 per group) mice. Metformin significantly decreased fasting blood glucose in PNA mice. E, GTT indicated no difference in glucose tolerance among groups (n = 10 per group). F, ITT showed no differences in insulin sensitivity (n = 10 per group). *, P < 0.05. ns, Not significant. ITT curves from untreated CON and PNA mice were generated from data reported previously (10).

Fig. 3.

Metformin improves estrous cycles in PNA mice. A and B, Representative estrous cycle plots from metformin (met)- and vehicle-treated CON (A) and PNA (B) mice. c1–c4 and p1–p4 refer to individual CON and PNA mouse numbers. Daily cycle stage designated as diestrus (D), proestrus (P), or estrus (E). C, Estrous cycle length was significantly longer in PNA mice before metformin but restored to CON levels after treatment (n = 10 per group). D, Percent of time in estrous was significantly lower in PNA than CON mice but no longer different after metformin. Different lowercase letters indicate groups with significantly different means. P < 0.05.

Fig. 4.

GnRH neurons from PNA mice have increased firing activity that is reversed by metformin. A, Representative graphs of firing rate over time in GnRH neurons from mice from each group. Events are binned in 60-sec intervals. B, Firing rate of GnRH neurons was higher in PNA mice (n = 17 cells from 10 mice) compared with CON (n = 17 cells from eight mice). Firing rate was lower in GnRH neurons from PNA mice treated with metformin (met) (n = 14 cells from seven mice) but was not different in cells from CON mice on metformin (n = 19 cells from eight mice). Percent quiescence (C) and maximum duration of quiescence (D) were lower in GnRH neurons from PNA mice not treated with metformin. Metformin restored quiescence measures of GnRH neurons from PNA mice but had no effect on CON mice. Quiescence is defined as 0–1 events/min. Different lowercase letters indicate groups with significantly different means. P < 0.05.

Fig. 5.

An AMPK antagonist stimulates firing in metformin-treated but not untreated mice. A, Representative plots of firing rate over time from each group. Black bar indicates time of CC application. CC induces an acute increase in firing in GnRH neurons from metformin (met)-treated mice. B, Percent of cells responding with a greater than 20% increase in firing rate in response to CC application. Significantly more cells from metformin-treated compared with untreated mice responded to the antagonist (P < 0.05). C, Mean percent change in firing rate in response to CC application (CON, n = 10 cells from five mice; PNA, n = 8 cells from three mice; CON metformin, n = 10 cells from four mice; and PNA metformin, n = 11 cells from five mice). Different lowercase letters indicate groups with significantly different means. P < 0.05.

Fig. 6.

Glucosensing is attenuated in GnRH neurons from metformin (met)-treated compared with untreated mice. A, Representative graphs of firing rate over time. Shaded region indicates period of low (0.2 mm) glucose application. Double-headed arrows in first graph indicate periods during which frequency was averaged for analysis. B, Firing rates in 5 mm glucose and during time intervals t1 and t2 after low-glucose application. Firing rate was significantly reduced by low glucose in GnRH neurons from untreated CON and PNA mice but not those treated with metformin (CON, n = 8 cells from five mice; PNA, n = 8 cells from four mice; CON metformin, n = 6 cells from three mice; and PNA metformin, n = 8 cells from three mice). *, P < 0.05 vs. 5 mm.

Fig. 7.

Serum reproductive hormone levels and uterine mass. A, LH levels were elevated in PNA mice. Metformin (met) restored LH in PNA mice to the level of CON mice (n = 9–14 per group). B and C, Testosterone and androstenedione levels were not different between PNA and CON mice; metformin increased testosterone but had no effect on androstenedione levels (n = 9–14 per group). D, Uterine mass was not different among groups (n = 19–23 per group). Different lowercase letters indicate groups with significantly different means. P < 0.05.