Ablating astrocyte insulin receptors leads to delayed puberty and hypogonadism in mice

Abstract

Insulin resistance and obesity are associated with reduced gonadotropin-releasing hormone (GnRH) release and infertility. Mice that lack insulin receptors (IRs) throughout development in both neuronal and non-neuronal brain cells are known to exhibit subfertility due to hypogonadotropic hypogonadism. However, attempts to recapitulate this phenotype by targeting specific neurons have failed. To determine whether astrocytic insulin sensing plays a role in the regulation of fertility, we generated mice lacking IRs in astrocytes (astrocyte-specific insulin receptor deletion [IRKOGFAP] mice). IRKOGFAP males and females showed a delay in balanopreputial separation or vaginal opening and first estrous, respectively. In adulthood, IRKOGFAP female mice also exhibited longer, irregular estrus cycles, decreased pregnancy rates, and reduced litter sizes. IRKOGFAP mice show normal sexual behavior but hypothalamic-pituitary-gonadotropin (HPG) axis dysregulation, likely explaining their low fecundity. Histological examination of testes and ovaries showed impaired spermatogenesis and ovarian follicle maturation. Finally, reduced prostaglandin E synthase 2 (PGES2) levels were found in astrocytes isolated from these mice, suggesting a mechanism for low GnRH/luteinizing hormone (LH) secretion. These findings demonstrate that insulin sensing by astrocytes is indispensable for the function of the reproductive axis. Additional work is needed to elucidate the role of astrocytes in the maturation of hypothalamic reproductive circuits.

Fig 1. Confirmation of astrocytic IR knockout model (IRKO^GFAP).

(A) Experimental study of cross-section of hypothalamus at Bregma −1.82 mm and Bregma −2.30 mm for IR^loxp and IRKO^GFAP (n = 3–4 per group) using TIRF and confocal microscopy (B) IF cross-sections (50 nm) of hypothalamus for IRKO^GFAP stained with GFAP and NeuN antibodies (n = 3–4 per group). (C) FACS dot plot showing the sorting gates for tdTomato^low/APC^low, tdTomato^low/APC^high, tdTomato^high/APC^low and tdTomato^high/APC^high from mice brain hypothalamic cells (n = 2 per group). RTPCR of hypothalamic gene expression levels of isolated astrocytes from FACS were reported as relative quantification (RQ = ^2-ΔΔCt) for IR^loxp, IRKO^GFAP and IRKO^GFAP nonastrocyte. IR^loxp (black bar), IRKO^GFAP (white bar), and IRKO^GFAP nonastrocyte (dashed white bar). Values are expressed as means ± SEM. *P < 0.05 IRKO^GFAP versus IR^loxp group. (D) Western blotting of protein expression from brain tissues were imaged and quantified for IR^loxp and IRKO^GFAP mice. IR^loxp (black bar) and IRKO^GFAP (white bar), (n = 4 per group, two brains pooled per lane). Values are expressed as means ± SEM. *P < 0.05 IRKO^GFAP versus IR^loxp group. The underlying data can be found in S1 Data. APC, allopycocyanin; FACS, fluorescence-activated cell sorting; GFAP, glial fibrillary acidic protein; IR; IRKO^GFAP, astrocyte-specific insulin receptor deletion; RQ, relative quantification.

Fig 2. Disruption in pubertal timing and adult fertility.

(A–C) Puberty onset was measured as balanopreputial separation in males and vaginal opening and first estrus in females. IR^loxp (black bar), IRKO^GFAP (white bar), n = 10–16 per group. (D) Female adult estrus cycle length and cell type analysis. P (predominantly nucleated cells), E (predominantly cornified epithelium cells), D (predominantly leukocytes), n = 10–13 per group. Values are expressed as means ± SEM. *P < 0.05 IRKO^GFAP versus IR^loxp group. (F–H) Percentage of matings for males resulting in pregnancy. Male litter sizes. Male interval from mating until birth of pups. IR^loxp (black bar) and IRKO^GFAP (white bar), n = 9–10 for males. Values are expressed as means ± SEM. *P < 0.05 IRKO^GFAP versus the IR^loxp group. (I–K) LH levels of males. FSH level of males. Testosterone levels of males. (n = 7–9 per group). IR^loxp (black bar) and IRKO^GFAP (white bar). Values are expressed as means ± SEM. *P < 0.05 IRKO^GFAP versus the IR^loxp group. (L–N) Percentage of matings for females resulting in pregnancy. Female litter sizes. Female interval from mating until birth of pups. IR^loxp (black bar) and IRKO^GFAP (white bar), n = 9 for females. Values are expressed as means ± SEM. *P < 0.05 IRKO^GFAP versus the IR^loxp group. (O–Q) LH levels of females. FSH level of females. Estradiol level of females. (n = 6–8 per group). IR^loxp (black bar) and IRKO^GFAP (white bar). Values are expressed as means ± SEM. *P < 0.05 IRKO^GFAP versus the IR^loxp group. The underlying data can be found in S1 Data. D, diestrus; E, estrus; FSH, follicle-stimulating hormone; IR, insulin receptor; IRKO^GFAP, astrocyte-specific insulin receptor deletion; LH, luteinizing hormone; P, proestrus.

Fig 3. Altered testes morphology and impaired spermatogenesis as well as ovarian morphology and follicle maturation at 6–7 months of age.

(A–B) Histological images of representative IR^loxp and IRKO^GFAP testes. (C–F) Analysis of number of spermatogonium, spermatocyte, spermatid, and spermatozoa of IR^loxp (n = 5) and IRKO^GFAP (n = 4). IR^loxp (black bar) and IRKO^GFAP (white bar). Values are expressed as means ± SEM. *P < 0.05 IRKO^GFAP versus the IR^loxp group. Histological images of IR^loxp (n = 4) and IRKO^GFAP (n = 3) female mice IR^loxp (black bar) and IRKO^GFAP (white bar). (I–M) Ovarian follicle maturation analysis of different follicle stages (primordial, primary, secondary, and Graafian) and corpora lutea in IR^loxp (n = 4) and IRKO^GFAP (n = 3) mouse ovaries. Values are expressed as means ± SEM. *P < 0.05 IRKO^GFAP versus the IR^loxp group. The underlying data can be found in S1 Data. IR, insulin receptor; IRKO^GFAP, astrocyte-specific insulin receptor deletion.

Fig 4. Altered PGES2 levels.

(A) Western blotting of PGES2 protein expression from FACS-isolated astrocytes were imaged and quantified for IR^loxp and IRKO^GFAP mice. IR^loxp (black bar) and IRKO^GFAP (white bar), (n = 4 per group, 2 brains pooled per lane). Values are expressed as means ± SEM. *P < 0.05 IRKO^GFAP versus IR^loxp group. The underlying data can be found in S1 Data. (B–C) Schematic diagram representing the mechanism of astrocyte modulation of HPG axis. FACS, fluorescence-activated cell sorting; HPG, hypothalamic pituitary gonadotropin; IR, insulin receptor; IRKO^GFAP, astrocyte-specific insulin receptor deletion; PGES2, prostaglandin E synthase 2.