Muscle-derived myostatin is a major endocrine driver of follicle-stimulating hormone synthesis

Abstract

Myostatin is a paracrine myokine that regulates muscle mass in a variety of species, including humans. In this work, we report a functional role for myostatin as an endocrine hormone that directly promotes pituitary follicle-stimulating hormone (FSH) synthesis and thereby ovarian function in mice. Previously, this FSH-stimulating role was attributed to other members of the transforming growth factor-β family, the activins. Our results both challenge activin's eponymous role in FSH synthesis and establish an unexpected endocrine axis between skeletal muscle and the pituitary gland. Our data also suggest that efforts to antagonize myostatin to increase muscle mass may have unintended consequences on fertility.

Fig. 1:. FSH synthesis is activin-independent in mice.

(A) Schematic representation of the proposed mode of activin B signaling in gonadotropes. The Xs represent the activin B knockout (KO) and type I receptor, ACVR1B, knockout models examined in Figure 1. Figure created in BioRender.com (BioRender.com/j95f951). (B) Schematic of mouse models used to assess activin signaling in gonadotropes, including wild-type (WT), control (floxed alleles only), Inhbb global KO, gonadotrope-specific Inhbb cKO, and gonadotrope-specific Acvr1b cKO mice. Serum (C) FSH and (D) LH levels (measured by multiplex ELISA) in wild-type or Inhbb KO mice. Pituitary gene expression assessed by RT-qPCR in (E) male and (F) female WT or Inhbb KO mice. Serum (G) FSH and (H) LH levels (measured by ELISA) in control or gonadotrope-specific Inhbb cKO mice. Serum (I) FSH and (J) LH levels (multiplex ELISA) in control or gonadotrope-specific Acvr1b cKO mice. Pituitary gene expression in (K) male and (L) female control or Acvr1b cKO mice. Animals were euthanized at 8 to 10 weeks old. Females were sampled at 7 am on estrus morning. Bar heights are group means. Each circle represents an individual mouse. Rpl19 was used as a housekeeping gene in E, F, K, and L. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. ns, non-significant.

Fig. 2:. FSH production depends on the type I receptor, TGFBR1, in gonadotropes.

(A) Schematic representation of the gonadotrope-specific TGFBR1 knockout model used in Figure 2. Figure created in BioRender.com (BioRender.com/x21t595). (B) Serum FSH (Luminex assay), (C) LH (ELISA), and pituitary gene expression (RT-qPCR) in (D) male and (E) female control (gray) and gonadotrope-specific Tgfbr1 cKO (purple) mice. Rpl19 was used as a housekeeping gene in D and E. (F) Antral follicle (AF) and corpora lutea (CL) counts from ovarian sections of 9- to 10-week-old control and Tgfbr1 cKO females (one ovary per mouse). (G) Number of pups per litter in 6-month breeding trials of control and Tgfbr1 cKO females. (H) Testicular weights (normalized to body weight) of control and Tgfbr1 cKO males. Bar heights are group means. Each circle represents an individual mouse. Females were sampled at 7 am on estrus morning. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. ns, non-significant.

Fig. 3:. Acvr1b/Tgfbr1 double cKO animals are FSH-deficient and hypogonadal.

(A) Schematic representation of the gonadotrope-specific Acvr1b/Tgfbr1 knockout model used in Figure 3. Figure created in BioRender (BioRender.com/s57z035). (B) Serum FSH and (C) LH levels (measured by ELISA), and pituitary gene expression (RT-qPCR) in (D) male and (E) female 9- to 10-week-old control (gray) and gonadotrope-specific Acvr1b/Tgfbr1 dcKO (maroon) mice. Control females were randomly cycling. Rpl19 was used as a housekeeping gene in D and E. (F) Number of pups per litter in 6-month breeding trials of control and Acvr1b/Tgfbr1 dcKO females. (G) Representative images of female reproductive tracts (top) and testes (bottom). Scale bar: 1 cm. (H) Ovarian weights (normalized to body weight). Bar heights are group means. Each circle represents an individual mouse. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. ns, non-significant. (I) Ovarian and (J) testicular histology sections stained with H&E. Boxed areas in the images at the left are expanded at the right. Scale bars: 200 μm.

Fig. 4:. Myostatin (MSTN) and GDF11 stimulate FSH synthesis.

(A) LβT2 cells were transfected with a murine Fshb promoter-luciferase (luc) reporter plasmid. Cells were then treated with medium or 0.5–2 nM GDF11 (orange) or MSTN (teal). Data represent mean ± SEM from 3 independent experiments (N=3). LβT2 cells were transfected with the Fshb-luc reporter and 5 nM of control, Acvr2a, Acvr1b, or Tgfbr1 siRNA. Cells were treated with medium or (B) GDF11 (1 nM) or (C) MSTN (2 nM). Bar heights are group means. Each circle represents an independent experiment. (D) Dot plots of Gdf11, Inhba, Inhbb, and Mstn expression in different cell lineages from snRNAseq of adult female and male murine pituitaries. (E) Schematic representation of the gonadotrope-specific Gdf11 and gonadotrope-specific Inhbb/Gdf11 double knockout mice used in Figure 4F and G. Figure created in BioRender.com (BioRender.com/o34c553). Serum FSH (measured by ELISA) in 9- to 10-week-old (F) control and Gdf11 cKO or (G) control and Inhbb/Gdf11 dcKO mice. Females were sampled at 7 am on estrus morning. Bar heights are group means. Each circle represents an individual mouse. (H) Schematic of intravenous (tail vein) injections of the indicated neutralizing antibodies in adult wild-type mice. (I) Percentage change in serum FSH levels 1, 2, 3, and 4 weeks following a single i.v. injection of mouse IgG or 5–20 mg/kg anti-MSTN/GDF11 antibody in adult male wild-type mice. The total amount of injected antibody was balanced to 20 mg/kg using mouse IgG. Data were normalized to FSH levels before injection for each animal. Data represent mean ± SEM (n=3/group). (J) Percentage change in serum FSH levels 2 or 7 days following a single i.v. injection of mouse IgG, anti-activin A, anti-MSTN, or anti-MSTN/GDF11 antibodies (10 mg/kg) in adult male wild-type mice. Data were normalized to FSH levels before injection. Data represent mean ± SEM (n=3/group). (K) Serum FSH levels (ELISA) and (L) number of eggs ovulated by female wild-type mice at natural ovulation following a single i.v. injection with the indicated neutralizing antibodies. Injected females were paired with wild-type males 7 days post-injection and samples collected on the morning of vaginal plugging. Bar heights are group means. Each circle represents an individual mouse. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. ns, non-significant.

Fig. 5:. Myostatin is a major driver of FSH synthesis and secretion in mice.

(A) Schematic representation of Mstn knockout or muscle-specific conditional KO (cKO) mice used in Figure 5B–K. Figure created in BioRender.com (BioRender.com/g88k582). (B) Serum FSH (ELISA) in 9- to 10-week-old WT/control (gray) and Mstn KO (teal) mice or (C) Mstn cKO (light blue) mice. Females were sampled at 7 am on estrus morning. (D) Number of eggs ovulated on estrus morning in 8- to 9-week-old WT/control and Mstn KO females or (E) Mstn cKO females. (F) Number of pups per litter in 3-month breeding trials of WT/control and Mstn KO or (G) Mstn cKO females. In B-G, bar heights are group means. Each circle represents an individual mouse. (H) Representative image of testes from a wild-type and a global Mstn KO mouse. Scale bar: 1 cm. (I) Schematic of intramuscular injections of control (AAV-control) or myostatin expressing adeno-associated viruses (AAV-MSTN) in Mstn KO mice. (J) Serum myostatin (MSTN) levels and (K) serum FSH levels (ELISA) 4 weeks after intramuscular injections of AAV-control or AAV-MSTN particles in 8-week-old male Mstn KO mice. Bar heights are group means. Each circle represents an individual mouse. (L) Schematic of i.v. injection of neutralizing antibodies in adult wild-type mice. (M) Percentage change in serum FSH levels 2 or 7 days following a single i.v. injection of human and mouse IgG, anti-activin A/B (15 mg/kg), and/or anti-MSTN/GDF11 antibodies (10 mg/kg) in adult male wild-type mice. The total amount of injected antibody was balanced across conditions using mouse IgG or human IgG. Data were normalized to FSH levels before injection for each mouse. Data represent mean ± SEM (n=3/group). (N) Pituitary Fshb expression (by RT-qPCR) in adult male mice one week after IgG or antibody injections. Bar heights are group means. Each circle represents an individual mouse. Rpl19 was used as a housekeeping gene. (O) Schematic of i.v. injection of antibodies in adult male Long-Evans rats (200–250 grams). (P) Percentage change in serum FSH levels 2 or 7 days following a single i.v. injection of mouse IgG or an anti-MSTN/GDF11 antibody (10 mg/kg) in male rats. Data were normalized to FSH levels before injection for each rat. Data represent mean ± SEM (n=3/group). * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. ns, non-significant.