Irisin deletion induces a decrease in growth and fertility in mice

Abstract

Background: Irisin, which is cleaved from fibronectin type III domain-containing protein 5 (Fndc5), plays an important role in energy homeostasis. The link between energy metabolism and reproduction is well known. However, the biological actions of irisin in reproduction remain largely unexplored.

Methods: In this study, we generated Fndc5 gene mutation to create irisin deficient mice. Female wild-type (WT) and Fndc5 mutant mice were fed with standard chow for 48 weeks. Firstly, the survival rate, body weight and fertility were described in mice. Secondly, the levels of steroid hormones in serum were measured by ELISA, and the estrus cycle and the appearance of follicles were determined by vaginal smears and ovarian continuous sections. Thirdly, mRNA-sequencing analysis was used to compare gene expression between the ovaries of Fndc5 mutant mice and those of WT mice. Finally, the effects of exogenous irisin on steroid hormone production was investigated in KGN cells.

Results: The mice lacking irisin presented increased mortality, reduced body weight and poor fertility. Analysis of sex hormones showed decreased levels of estradiol, follicle-stimulating hormone and luteinizing hormone, and elevated progesterone levels in Fndc5 mutant mice. Irisin deficiency in mice was associated with irregular estrus, reduced ratio of antral follicles. The expressions of Akr1c18, Mamld1, and Cyp19a1, which are involved in the synthesis of steroid hormones, were reduced in the ovaries of mutant mice. Exogenous irisin could promote the expression of Akr1c18, Mamld1, and Cyp19a1 in KGN cells, stimulating estradiol production and inhibiting progesterone secretion.

Conclusions: Irisin deficiency was related to disordered endocrinology metabolism in mice. The irisin deficient mice showed poor growth and development, and decreased fertility. Irisin likely have effects on the expressions of Akr1c18, Mamld1 and Cyp19a1 in ovary, regulating the steroid hormone production. This study provides novel insights into the potential role of irisin in mammalian growth and reproduction.

Keywords: Growth and development; Hormone metabolism; Irisin; Reproduction.

Fig. 1

Generation of Fndc5 mutation mice. a: Schematic representation of the gene targeting procedure. The black arrowhead indicates the target exon. b: Sequences of the Fndc5 mRNA transcripts in mice and the proteins predicted to be encoded by the respective mRNAs. c: Confirmation of Fndc5 mutation. Genotyping (left panel) and Western blot (right panel) analyses were consistent with successful Fndc5 mutation

Fig. 2

Irisin deletion influenced the survival and body weight of Fndc5 mutant mice. a: The Fndc5 mutant mice showed high mortality. Percentages of surviving WT (n = 44) and Fndc5 mutant (n = 51) mice (P < 0.0001). b: Appearance of pups. c: Appearance of adult mice. d: Body weight (n_wt = 39_, n_mutant = 20). e, F: Growth hormone (GH) and insulin-like growth factor I (IGF-1) concentrations (n = 12 per group); *P < 0.05 compared to WT mice

Fig. 3

Irisin deletion affected the estrous cycle in Fndc5 mutant mice. a: Example images of estrous cycle phases. Yellow, red, and blue arrowheads indicate epithelial nucleated cells, squamous cells, and leucocytes, respectively; 200x. b: Representative estrous cycle of the mice in each group (n = 10). c: The distribution of estrous cycle phases (n = 10). *P < 0.05 compared to WT mice

Fig. 4

Ovary morphological analysis. a-c: Morphological findings for the ovaries. Ovarian mass index = ovary weight (g) / body weight (g); d: The primordial follicle to antral follicle ratio determined by in vivo ovarian analysis (n = 10). e: The antral follicle to total follicle ratio (n = 10). f: H&E staining of follicles in different stages in WT and Fndc5 mutant mice. Black arrowheads, primordial follicle; yellow arrowheads, primary follicle; red arrowheads, antral follicle. *P < 0.05 compared to WT mice

Fig. 5

Hormone concentrations in Fndc5 mutant mice (n = 10 per group). a: Estradiol (E₂). b: Progesterone (P). c: Testosterone (T). d: Follicle-stimulating hormone (FSH). e: Luteinizing hormone (LH). *P < 0.05 compared to WT mice

Fig. 6

Effects of irisin on hormone production in KGN cells. Cells were incubated with increasing concentrations of irisin (10–30 nM) or si-Fndc5 (100 nM). a: Irisin stimulated the phosphorylation of the p38 and ERK proteins. b: The E₂ concentration in the conditioned culture medium was measured by a specific radioimmunoassay. c: The progesterone (P) concentration was measured. Data are presented as the mean ± SD, (n = 3 per group); *P < 0.05 compared to the control group

Fig. 7

mRNA expression related to reproduction is downregulated in Fndc5 mutant mice. a: The heat map for mRNA analyses of the ovaries, muscle and hypothalamus in WT and Fndc5 mutant mice. b: The downregulated mRNAs in Fndc5 mutant mice are associated with steroid hormone synthesis. c: The different MS/MS spectra of Akr1c18, Mamld1 and Cyp19a1 in Fndc5 mutant and WT mice are shown. d: qRT-PCR results for the ovaries confirmed the decreased expression of Akr1c18, Mamld1 and Cyp19a1 in Fndc5 mutant mice compared with WT mice. (n = 5 biologically independent Fndc5 mutant or WT mice). Data are presented as the mean ± SD; *P < 0.05 compared to WT mice

Fig. 8

The effects of irisin on the expression of Akr1c18, Mamld1 and Cyp19a1 in vitro. KGN cells were cultured in the absence or presence of irisin (10–30 nM) for 48 h. a: Irisin increased the mRNA levels of Akr1c18, Mamld1 and Cyp19a1 (data from qRT-PCR). The mRNA levels were normalized to the GAPDH mRNA levels. Data are presented as the mean ± SD (n = 3). b, c: After exposure of KGN cells to the indicated concentrations of irisin for 72 h, the protein levels of Akr1c18, Mamld1 and Cyp19a1 were determined by Western blot analysis, and protein expression levels were quantified. d: mRNA was detected 48 h after siRNA transfection. e, f: Fndc5, Akr1c18, Mamld1 and Cyp19a1 expression at the protein level was detected 72 h after transfection (n = 3); *P < 0.05 compared to the control group