Developmental Programming: Insulin Sensitizer Prevents the GnRH-Stimulated LH Hypersecretion in a Sheep Model of PCOS

Abstract

Prenatal testosterone (T) treatment recapitulates the reproductive and metabolic phenotypes of polycystic ovary syndrome in female sheep. At the neuroendocrine level, prenatal T treatment results in disrupted steroid feedback on gonadotropin release, increased pituitary sensitivity to GnRH, and subsequent LH hypersecretion. Because prenatal T-treated sheep manifest functional hyperandrogenism and hyperinsulinemia, gonadal steroids and/or insulin may play a role in programming and/or maintaining these neuroendocrine defects. Here, we investigated the effects of prenatal and postnatal treatments with an androgen antagonist (flutamide [F]) or an insulin sensitizer (rosiglitazone [R]) on GnRH-stimulated LH secretion in prenatal T-treated sheep. As expected, prenatal T treatment increased the pituitary responsiveness to GnRH leading to LH hypersecretion. Neither prenatal interventions nor postnatal F treatment normalized the GnRH-stimulated LH secretion. Conversely, postnatal R treatment completely normalized the GnRH-stimulated LH secretion. At the tissue level, gestational T increased pituitary LHβ, androgen receptor, and insulin receptor-β, whereas it reduced estrogen receptor (ER)α protein levels. Although postnatal F normalized pituitary androgen receptor and insulin receptor-β, it failed to prevent an increase in LHβ expression. Contrarily, postnatal R treatment restored ERα and partially normalized LHβ pituitary levels. Immunohistochemical findings confirmed changes in pituitary ERα expression to be specific to gonadotropes. In conclusion, these findings indicate that increased pituitary responsiveness to GnRH in prenatal T-treated sheep is likely a function of reduced peripheral insulin sensitivity. Moreover, results suggest that restoration of ERα levels in the pituitary may be one mechanism by which R prevents GnRH-stimulated LH hypersecretion in this sheep model of polycystic ovary syndrome-like phenotype.

Figure 1.

Schematic showing the temporal sequence of experimental procedures. In the first study (upper panel), female sheep were treated prenatally (GD30–GD90) with vehicle (C, n = 7 female offspring), T (n = 6 female offspring), TF (n = 7 female offspring), or TR (n = 7 female offspring). Additionally, 2 groups of females prenatally treated with T were subjected to postnatal intervention with either F (T+F, n = 6 female offspring) or R (T+R, n = 8 female offspring). Postnatal treatments started at approximately 8 weeks of age and continued throughout the study. Pituitary sensitivity to exogenous GnRH was tested in vivo at approximately 15 months of age during the seasonal anestrous period. At approximately 24 months of age, ewes were euthanized and pituitary tissues were harvested, fixed in paraformaldehyde, and processed for immunohistochemistry (IHC). Female offspring (C, n = 8; T, n = 10; T+F, n = 9; T+R, n = 9) in study 2 (bottom panel) were subjected to similar experimental procedures, with the exception that prenatal T treatment spanned from GD60 to GD90 (as opposed to GD30–GD90 in study 1). At approximately 3 years of age, females were euthanized, and pituitary tissues were harvested, snap frozen, and processed for Western blot analysis (WB). No TF or TR groups were generated in study 2.

Figure 2.

Effects of gestational T excess and pharmacological intervention with an androgen antagonist (F) or an insulin sensitizer (R) on the GnRH-stimulated LH secretion in female sheep. A, B, and D, Two representative LH profiles of females treated with prenatal vehicle (C, n = 7), prenatal T (T, n = 6), prenatal T and prenatal F (TF, n = 7), prenatal T and prenatal R (TR, n = 7), prenatal T plus postnatal F (T+F, n = 6), or prenatal T plus postnatal R (T+R, n = 8). Arrowheads indicate time of GnRH injections. Bar graphs depict mean (±SEM) LH pulse peak and pulse amplitude of prenatal intervention (C) and postnatal intervention groups (E). Please note that, to facilitate comparison between groups, mean LH pulse peak and amplitude for C and T groups are repeated in C and E. Means with different superscripts are significantly (P < .05) different.

Figure 3.

Effects of gestational T excess and postnatal intervention with an androgen antagonist (F) or an insulin sensitizer (R) on the protein expression of LHβ and key regulators of LH synthesis/secretion in the pituitary from female sheep. Representative Western blottings and mean (±SEM) protein level ratio of LHβ to GAPDH (A), GnRH-R to GAPDH (B), AR to GAPDH (C), ERα to GAPDH (D), IRβ to GAPDH (E), and PTEN to GAPDH (F) in the anterior pituitary from females treated with prenatal vehicle (C, n = 8), prenatal T (T, n = 10), prenatal T plus postnatal F (T+F, n = 9), or prenatal T plus postnatal R (T+R, n = 9). Means with different superscripts are significantly (P < .05) different.

Figure 4.

Effects of gestational T excess and postnatal intervention with an androgen antagonist (F) or an insulin sensitizer (R) on insulin signaling in the pituitary from female sheep. Representative Western blottings and mean (±SEM) protein level ratio of p-AKT to AKT, AKT to GAPDH (A), p-mTOR to mTOR, mTOR to GAPDH (B), p-ERK to ERK, and ERK to GAPDH (C) in the anterior pituitary from females treated with prenatal vehicle (C, n = 8), prenatal T (T, n = 10), prenatal T plus postnatal F (T+F, n = 9), or prenatal T plus postnatal R (T+R, n = 9). A treatment effect (P = .04) was observed for the ratio of p-mTOR to mTOR; however, none of the group comparisons reached statistical significance in the post hoc analysis (Tukey's HSD test).

Figure 5.

Effects of gestational T excess and pharmacological intervention with an androgen antagonist (F) or an insulin sensitizer (R) on LHβ and ERα immunoreactivity in the pituitary from female sheep. Confocal microscope images (1-μm optical sections) depicting LHβ (green), ERα (red), and DAPI (blue) staining in the anterior pituitary from females treated with prenatal vehicle (C, n = 7), prenatal T (T, n = 6), prenatal T and prenatal F (TF, n = 7), prenatal T and prenatal R (TR, n = 7), prenatal T plus postnatal F (T+F, n = 6), or prenatal T plus postnatal R (T+R, n = 8). White arrows indicate LHβ-immunoreactive cells that colocalize with ERα, and yellow arrow illustrates an LHβ-immunoreactive cell that does not colocalize with ERα. Bar graphs demonstrate the mean (±SEM) number of LHβ-immunoreactive cells (A), number of ERα-immunoreactive cells (B), and percentage of LHβ-immunoreactive cells that colocalize with ERα (C). Scale bar, 50 μm. Means with different superscripts are significantly (P < .05) different.

Figure 6.

Schematic drawing of the proposed events leading to pituitary LH hypersecretion in prenatal T-treated sheep. Adult sheep prenatally exposed to T excess manifest reduced insulin sensitivity in the liver and skeletal muscle resulting in peripheral insulin resistance and compensatory hyperinsulinemia. In turn, increased circulating concentrations of insulin stimulate the anterior pituitary (AP), which remains insulin sensitive, to synthesize and secrete large amounts of LH. Although the exact mechanisms by which insulin promotes LH hypersecretion in prenatal T-treated sheep remain unclear, a reduction in ERα levels in the AP appears to be involved.