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. 2013 Jun;123(6):2421-33.
doi: 10.1172/JCI65952.

Osteocalcin Regulates Murine and Human Fertility Through a Pancreas-Bone-Testis Axis

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Osteocalcin Regulates Murine and Human Fertility Through a Pancreas-Bone-Testis Axis

Franck Oury et al. J Clin Invest. .
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Abstract

The osteoblast-derived hormone osteocalcin promotes testosterone biosynthesis in the mouse testis by binding to GPRC6A in Leydig cells. Interestingly, Osteocalcin-deficient mice exhibit increased levels of luteinizing hormone (LH), a pituitary hormone that regulates sex steroid synthesis in the testes. These observations raise the question of whether LH regulates osteocalcin's reproductive effects. Additionally, there is growing evidence that osteocalcin levels are a reliable marker of insulin secretion and sensitivity and circulating levels of testosterone in humans, but the endocrine function of osteocalcin is unclear. Using mouse models, we found that osteocalcin and LH act in 2 parallel pathways and that osteocalcin-stimulated testosterone synthesis is positively regulated by bone resorption and insulin signaling in osteoblasts. To determine the importance of osteocalcin in humans, we analyzed a cohort of patients with primary testicular failure and identified 2 individuals harboring the same heterozygous missense variant in one of the transmembrane domains of GPRC6A, which prevented the receptor from localizing to the cell membrane. This study uncovers the existence of a second endocrine axis that is necessary for optimal male fertility in the mouse and suggests that osteocalcin modulates reproductive function in humans.

Figures

Figure 1
Figure 1. Analysis of the rescue of male fertility phenotype in Lhb–/– male mice after osteocalcin injections.
(A) Measurement of the uncarboxylated (GLU-OCN), carboxylated (GLA13-OCN), total (total-OCN), and undercarboxylated (GLU13-OCN) forms of osteocalcin in the serum of 10-week-old Lh-deficient mice (Lhb–/–) (n = 7) versus WT (n = 5). (B) Circulating testosterone levels in 12-week-old WT (n = 5) and Lhb–/– (n = 7) mice and in Lhb–/– mice injected for 1 month with PBS (n = 8) or osteocalcin (3 ng/ml) (n = 5). (C and D) Testes cross-sections of WT and Lhb–/– injected for 1 month with PBS, osteocalcin (3 ng/g/d) or hCG (5 UI twice a week). (C) Histological demonstration of the interstitial tissue hypoplasia (2 first rows) and of the absence of spermatogenesis (2 last rows) observed in Lhb–/– mice. While injections of hCG rescue the phenotype, PBS or osteocalcin do not. The black line delimits the frame zoom shown in the second row. The head arrows point to the interstitial tissue containing Leydig cells. Rsp, round spermatids; Esp, elongated spermatids. (D) Immunofluorescence, using anti-Cyp17, anti-3β-HSD, or anti-Cyp11a antibodies as markers of mature Leydig cells. (E) Testis size and (F) testis weight normalized to BW (mg/g of BW) in Lhb–/– mice injected with PBS (n = 8), osteocalcin (n = 4), or hCG (n = 3) compared with WT mice. All analyses were performed on nonbreeder C57BL/6J mice. Scale bar: 100 μm. *P < 0.05; **P < 0.01.
Figure 2
Figure 2. Analysis of Osteocalcin–/– male mouse fertility after hCG or osteocalcin injection.
Sperm count (A); testis (B), epididymal (C), and seminal vesicle (D) weights normalized to BW (mg/g of BW), (E) circulating testosterone levels in WT and Osteocalcin–/– mice at 10 weeks of age, both injected either with PBS (WT, n = 20; Osteocalcin–/–; n = 14; Gprc6a–/–, n = 7), hCG (WT, n = 12; Osteocalcin–/–, n = 8; Gprc6a–/–, n = 6) (5 UI, twice a week), or osteocalcin (WT, n = 6; Osteocalcin–/–, n = 6; Gprc6a–/–, n = 6) (3 ng/g/d). All analyses were performed on nonbreeder 129/Sv mice. Error bars represent SEM. *P < 0.05, Student’s t test. Specifically, P < 0.05 versus WT injected with hCG (5 UI, twice a week); and P < 0.05 versus WT injected with osteocalcin (3 ng/g/d).
Figure 3
Figure 3. The osteocalcin reproductive function is hampered in the absence of proper bone resorption.
(A) Schematic representation of the strategy used to generate Ctsk-Cre;DTAfl/+ male mice. (BD) Histological and histomorphometric analyses of vertebrae in control (n = 8) and Ctsk-Cre;DTAfl/+ (n = 9) male mice 4 months after transplantation. (B) Von Kossa/Van Giesen staining. Bone volume over trabecular volume (BV/TV%) is indicated below the pictures. (C) TRAP staining to reveal osteoclasts. Osteoclast surface per bone surface (Oc.S/BS [%]) and number of osteoclasts per bone trabecular surface (N. Oc/Bpm [1 mm]) are indicated below the pictures. (D) Toluidine blue staining showing an important presence of cartilage remnants (indicated by the asterisk) in Ctsk-Cre;DTAfl/+ male mice versus WT. (E) Measurement of uncarboxylated, carboxylated, total, and undercarboxylated forms of osteocalcin in the serum of 10-week-old Ctsk-Cre;DTAfl/+ versus WT male mice. (F) Sperm counts and (G) circulating testosterone levels; (HJ) testis, epididymal, and seminal vesicle weights normalized to BW (mg/g of BW) in Ctsk-Cre;DTAfl/+ (n = 12) versus WT (n = 11) male mice. (K) Circulating LH measurement in control and Ctsk-Cre;DTAfl/+ mice. (L) qPCR analysis of the expression of steroidogenic acute regulatory protein (StAR), cholesterol side-chain cleavage enzyme (Cyp11a), cytochrome P-450 17 α (Cyp17), 3-β-hydroxysteroid dehydrogenase (3β-HSD), aromatase enzyme (Cyp19), and 17- β-hydroxysteroid dehydrogenase (HSD-17) in Ctsk-Cre;DTAfl/+ (n = 12) compared with WT (n = 11) male mice. All analyses presented were performed on nonbreeder C57BL/6J mice. Scale bars: 200 μm). *P < 0.05; **P < 0.01; ***P < 0.001.
Figure 4
Figure 4. An increase in osteoclast number favors both osteocalcin activity and male fertility.
(A) Measurement of the uncarboxylated, carboxylated, total, and undercarboxylated forms of osteocalcin in serum of WT (n = 12), Opg–/– (n = 10), and Opg+/– mice (n = 10). (B) Sperm count; (C) testis, (D) epididymal; and (E) seminal vesicle weights normalized to BW (mg/g of BW). (F) Circulating testosterone levels in WT (n = 12), Opg–/– (n = 10), Opg+/– (n = 10), and Osteocalcin+/–; Opg+/– (n = 6) mice at 12 weeks of age. (G) qPCR analysis of the expression of StAR, Cyp11a, Cyp17, 3β-HSD, Cyp19, and HSD-17 in Opg–/– (n = 10) and Opg+/– (n = 10) compared with WT (n = 12) male mice. (H) Circulating LH measurement in Opg–/– (n = 10) and Opg+/– (n = 10) compared (%) with WT (n = 12) male mice. All analyses presented were performed on nonbreeder C57BL/6J mice. *P < 0.05; **P < 0.01; ***P < 0.001.
Figure 5
Figure 5. Insulin signaling in osteoblasts favors testosterone production.
(A) Testis size, and (B) testis, (C) epididymal, and (D) seminal vesicle weights normalized to BW (mg/g of BW); (E) sperm count; (F) circulating testosterone levels in InsRosb–/– versus WT male mice. (G) qPCR analysis of the expression of StAR, Cyp11a, Cyp17, 3β-HSD, Cyp19, and HSD-17 genes in testes of InsRosb–/– (n = 10) and WT (n = 12) mice. All analyses presented were performed on nonbreeder mix background (129/Sv: 87.5%; 129/Sv: 12.5%) mice. *P < 0.05; **P < 0.01.
Figure 6
Figure 6. Insulin signaling in osteoblasts promotes male fertility in an osteocalcin-dependent manner.
(A) Sperm counts, (BD) testis, epididymal, and seminal vesicle weights normalized to BW (mg/g of BW); (E) circulating testosterone levels in InsRosb+/– (n = 5), InsRosb+/–;Osteocalcin+/– (n = 6), and InsRosb+/–; Gprc6a+/– (n = 6) versus control (n > 8) male mice. All analyses presented were performed on nonbreeder mix background (129/Sv: 75%; C57BL/6J: 25%) mice. *P < 0.05; **P < 0.01.
Figure 7
Figure 7. Identification of an amino acid substitution of GPRC6A associated with decreased fertility in humans.
(A) T→A missense mutation in GPRC6A seen in 2 patients. (B) Localization of the F464Y substitution-mutation in GPRC6A. (C) Immunofluorescence of HEK293T cells transfected with WT (GPRC6A-MYC) or mutant (GPRC6A-MYC-F464Y) GPRC6A. Nuclei are stained with DAPI (blue), MYC-tag in green, and the membrane marker α 1 sodium potassium ATPase in red. (D) cAMP production upon osteocalcin stimulation (3 ng/ml) in HEK293T cells transfected with control (MYC-pcDNA3), WT (GPRC6A-pcDNA3), mutant (GPRC6A-mutant-pcDNA3), or both forms of GPRC6A. (E) cAMP production upon osteocalcin stimulation (3 ng/ml) in HEK293T cells transfected with WT (GPRC6A-pcDNA3), mutant (GPRC6A-mutant-pcDNA3) or both forms of GPRC6A (ratio of 1[WT]: 0.1 [mutant], 1:0.25, 1:0.5, 1:0.75, and 1:1). (F) qPCR and Western blot analysis of HEK293T cells cotransfected with WT (GPRC6A-FLAG) and mutant (GPRC6A-MYC-F464Y) GPRC6A at different ratios. (G and H) Immunofluorescence analyses of HEK293T cells cotransfected with WT and mutant GPRC6A at a ratio of (G) 1:1 (WT/mutant) and (H) 1:0.25. FLAG-tag stained in red, MYC-tag in green, and nuclei in blue (DAPI). (I) qPCR analysis of StAR expression in TM3 cells infected with pLenti6.3/V5 lentiviral vectors containing WT GPRC6A or the F464Y mutant and treated with vehicle or osteocalcin (1 ng/ml). (J) qPCR analysis of StAR, 3β-HSD, and Cyp11a expression in WT testis injected with vehicle, WT GPRC6A (LV-GPRC6A-WT), or the GPRC6A-F464Y-mutant (LV-GPRC6A-F464Y) (n = 4 for each conditions). Scale bar: 10 μm. *P < 0.05; ***P < 0.001.

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