The effect of insulin signaling on female reproductive function independent of adiposity and hyperglycemia

Abstract

Physiological states of insulin resistance such as obesity and diabetes have been linked to abnormalities in female reproductive function. However, it is difficult to distinguish the direct effects of impaired insulin signaling from those of adiposity or hyperglycemia because these conditions often coexist in human syndromes and animal models of insulin resistance. In this study, we used lean, normoglycemic mouse lines with differing degrees of hyperinsulinemia and insulin receptor (Insr) expression to dissect the effects of altered insulin signaling on female reproduction. All three mouse lines [Ttr-Insr(-/-), Insr(+/-), and Insr(+/+) (wild type)] are able to maintain fertility. However, the insulin-resistant and hyperinsulinemic mice demonstrate altered duration of estrous cycles as well as aberrant distribution and morphology of ovarian follicles. These effects appear to be independent of hyperandrogenism in the mice. Pregnancy studies indicate decreased success in early progression of gestation. In successful pregnancies, decreased embryo weights and increased placental calcification also implicate altered insulin signaling in later gestational effects. Thus, abnormal insulin signaling, independent of adipose tissue mass, adipokine expression levels, and hyperglycemia, can affect parameters of the female hypothalamic-pituitary-gonadal axis and pregnancy outcomes.

Figure 1

Metabolic analyses. The data represent the mean ± sem fasted and fed glucose (A) (n = 10–30) and insulin levels (B and C) (n = 6–10) in WT (open bar), IR^+/− (striped bar), and L1 (closed bar) females. *, P < 0.01; **, P < 0.001. D, Intraperitoneal glucose tolerance test. The mean glucose level ± sem at each time point after glucose injection is presented. Open diamond, WT; closed square, IR^+/−; closed triangle, L1. E and F, The mean ± sem weight and percent body fat in 2- and 3-month-old WT, IR^+/−, and L1 females (n = 6–20). G and H, Adiponectin and leptin expression levels were assessed with nonfasting samples (n = 7–15).

Figure 2

Levels of insulin receptor expression in the ovary (A), brain (B), and uterus (C). Single brain and uterus samples were used to generate protein lysates; pooled samples from five animals were used for the ovarian lysate. Immunoblot panels represent expression levels of either insulin receptor (Insr) or the loading control protein (actin). The histograms present the ratio of Insr to actin expression.

Figure 3

Assessment of ovarian function. A, Estrous cycling. The data represent the average number of days ± sem per estrous cycle (n = 6–11). B, Follicular analysis. The average number of healthy primordial, primary, preantral, antral, and graffian follicles (n = 6–8) is shown. C, Superovulation. The number of oocytes retrieved ± sem is shown (n = 7–9). *, P < 0.05. Polyovular follicles in IR^+/− ovaries (indicated with white arrows). Photomicrographs of primary (D), preantral (E), and antral (F) follicles are depicted. For thecal dimensions, the mean ± sem of thecal thickness (TT, total thickness; TI, theca interna) in WT, IR^+/−, polyovular IR^+/− (IR^+/− POL), and L1 follicles is presented (G). Granulosa cell morphology in WT (H) and a subset of IR^+/− ovaries (I) is shown.

Figure 4

Gestational analyses and metabolic characterization. A, Fed glucose levels from gestational d 0, 9.5, 14.5, and 17.5 remained in the nondiabetic range in WT (closed square), IR^+/− (open square), and L1 (closed triangle) mice. B, The data represent the mean ± sem fed insulin level in late gestation, d 17.5 (n = 5–8). *, P < 0.05; **, P < 0.005. C, Pregnancy outcomes and unsuccessful matings. The data represent the percentage of plugged females that did not proceed to successful pregnancies (n = 7–10). D, Placental histology. Histological sections of placentae from gestational d 17.5 were evaluated with light microscopy. Percentage of placental diameter consisting of labyrinthian (L) and spongiform (S) layers is presented. Foci of calcifications in IR^+/− (E) and L1 (F) placentae are indicated with black arrows. Photomicrographs were taken under ×20 magnification.