Osteocalcin and LH define 2 modes of regulating male fertility in the mouse.
The main endocrine pathway regulating male fertility is the hypothalamo-pituitary axis, in which LH, a heterodimer between an α-subunit common to several peptide hormones and a β-subunit specific to LH, favors testosterone biosynthesis (7
). Although less severe, the reproductive phenotype of Osteocalcin–/–
male mice bears some resemblance to the one seen in Lhb–/–
(LH-deficient) male mice, as they are both characterized by a defect in testosterone synthesis and testosterone-dependent events (2
). However, LH circulating levels are high in Osteocalcin–/–
male mice. These observations prompted us to ask whether LH may regulate osteocalcin reproductive function.
If this were the case, one would expect that circulating levels of the undercarboxylated (active) form of osteocalcin should be low in Lhb–/–
male mice and that injections of osteocalcin would rescue, completely or at least in part, the hypotestosteronemia of Lhb–/–
male mice. However, circulating levels of undercarboxylated (GLU13) osteocalcin were not lower in Lhb–/–
than in WT male mice (Figure A), and daily injections of osteocalcin for 1 month in 6-week-old Lhb–/–
male mice did not normalize circulating testosterone levels (Figure B). Moreover, histological analysis of testes of 10-week-old Lhb–/–
male mice injected with osteocalcin failed to show any improvement in spermatogenesis or a reversal of their Leydig cell hypoplasia (Figure , C and D, and Supplemental Figure 1A; supplemental material available online with this article; doi:
). The dose of osteocalcin used in this experiment was chosen because it is sufficient to increase testosterone synthesis by Leydig cells in culture (2
). Immunofluorescence analysis of mature Leydig cells using anti-Cyp17, anti–3β-HSD or anti-Cyp11a antibodies showed that daily injections of osteocalcin for 30 days did not increase the number of Leydig cells in Lhb–/–
male mice, while injections of the placental homolog of LH, human chorionic gonadotropin (hCG), did (Figure D and Supplemental Figure 1A). Therefore, and unlike what was achieved by hCG injections, osteocalcin injections did not increase testis size and weight, Leydig cell count, or spermatogenesis in Lhb–/–
male mice (Figure , E and F). This was not due to a poor bioactivity of the recombinant protein, since the same preparation of osteocalcin corrected all reproductive abnormalities seen in Osteocalcin–/–
mice but not in Gprc6a–/–
mice (Figure ). To rule out that the failure of the osteocalcin injections to increase testis weight was due to the fact that they were performed in adult mice, we repeated this experiment in 10-day-old Lhb–/–
mice that were injected for 30 days with either hCG (5 UI, twice a week) or osteocalcin (3 ng/g of BW, daily). Here again, while hCG treatment increased Leydig cell number, osteocalcin did not (Supplemental Figure 1B).
Analysis of the rescue of male fertility phenotype in Lhb–/– male mice after osteocalcin injections.
Analysis of Osteocalcin–/– male mouse fertility after hCG or osteocalcin injection.
To further distinguish between LH-dependent and osteocalcin-dependent regulation of male fertility, we performed the reverse experiment. Namely, we injected 6-week-old Osteocalcin–/– or Gprc6a–/– male mice with hCG (5UI, twice a week) for 1 month, reasoning that if osteocalcin were a mediator of LH reproductive function, these injections would not improve the reproductive abnormalities seen in Osteocalcin–/– mice. Instead, this treatment normalized all parameters analyzed: testes, epididymal, and seminal vesicle weights, sperm count, and testosterone circulating levels in Osteocalcin–/– male mice. Finally, circulating LH level was equally decreased in WT and Osteocalcin–/– serum after treatment with hCG (Supplemental Figure 1C). Taken together, these experiments suggest that LH promotes testosterone biosynthesis in an osteocalcin-independent manner.
Osteocalcin and LH do not regulate each other.
In the next set of experiments, we asked whether osteocalcin regulates Lh expression and/or whether LH regulates Osteocalcin expression. For Osteocalcin to regulate Lh expression it would first require that its receptor, Gprc6a, is expressed in the hypothalamus or the pituitary gland.
To address this issue, we first studied the Gprc6a endogenous pattern of expression of by in situ hybridization. While expression of this gene could be detected in Leydig cells, it was undetectable in the hypothalamus and pituitary gland. Concerned that this technique was not sensitive enough, we then used a second and more sensitive assay, quantitative PCR (qPCR), to measure Gprc6a expression. As an internal negative control of specificity, we also used in this experiment tissues originating from Gprc6a–/– mice. This assay failed to detect any expression of Gprc6a in the hypothalamus and pituitary gland in WT mice above what was seen in Gprc6a–/– mice (Supplemental Figure 2, A and B). We verified that the primers used could detect Gprc6a expression in WT but not Gprc6a–/– testes (Supplemental Figure 2B). Taken together, these 2 different experiments indicate that the regulation of testosterone synthesis by osteocalcin in Leydig cells cannot be ascribed to a measurable influence of osteocalcin signaling through Gprc6a in the hypothalamus or the pituitary gland.
Conceivably, however, LH could be required for osteocalcin stimulation of testosterone biosynthesis by Leydig cells. Evidence from 2 experiments suggested that this is not the case. First, the positive effect of osteocalcin on testosterone synthesis in Leydig cells was recorded when cells were maintained in serum-free medium, i.e., in the total absence of LH (2
). Second and more directly, in cell culture, hCG does not regulate expression of Osteocalcin
or of genes modifying it in osteoblasts (Supplemental Figure 3, A–C). Hence, taken collectively, results presented in Figures and suggest the notion that osteocalcin regulates male fertility independently of the hypothalamo-pituitary-axis; they also failed to provide any evidence that LH regulates Osteocalcin
Bone resorption as a determinant of osteocalcin reproductive function in the mouse.
By dissociating pituitary-dependent from bone-dependent regulation of male fertility, the experiments presented above raised the question of the identity of upstream regulators of osteocalcin reproductive function.
That the ability of osteocalcin to favor glucose homeostasis is determined by osteoclastic bone resorption prompted us to ask whether male fertility was another physiological function to be added to the credit of bone resorption (16
). To address this question, we relied on 2 mouse models, a loss-of-function model and a gain-of-function model.
First, we generated a mouse model demonstrating a profound depletion of the mature osteoclasts population by crossing Ctsk-Cre
mice, in which the Cre recombinase is expressed in mature osteoclasts only (17
), with DTAfl/+
mice that express a flox-stop-flox diphtheria toxin subunit α gene (DTA) cassette under the control of the Rosa26
mice are phenotypically WT because the flox-stop-flox cassette present in front of the DTA
gene prevents expression of the DTA protein in any tissues. However, Cre-mediated removal of the stop cassette in osteoclasts only, in Ctsk-Cre
double knockin mice, leads to osteoclast-specific expression of DTA, a toxic protein, and osteoclast death (Figure A).
The osteocalcin reproductive function is hampered in the absence of proper bone resorption.
Ctsk-Cre;DTAfl/+ mice developed a classical osteopetrosis phenotype characterized by very dense bones, as seen in x-rays, and the absence of incisor eruption because of a severe impairment in bone resorption (Supplemental Figure 4A). All mutant animals died between 2 and 3 weeks of age. To circumvent this postnatal lethality, we transplanted Ctsk-Cre;DTAfl/+ fetal liver HSCs into WT irradiated adult mice; since osteoclasts are of hematopoietic origin, mice transplanted with Ctsk-Cre;DTAfl/+ HSCs should be depleted of osteoclasts. Four months after transplantation, bone and fertility phenotypes of these animals were analyzed.
Bone histomorphometry verified that Ctsk-Cre
transplanted mice developed a high bone-mass phenotype due to a 15-fold decrease in the number of osteoclasts (Figure , B and C). The presence of cartilage remnants characterized this osteopetrosis phenotype (Figure D). These osteopetrotic mice showed more than a 2-fold decrease in the undercarboxylated (GLU13) form of osteocalcin (Figure E). This is more severe than what is seen in Osteocalcin+/–
transplanted mice also demonstrated a significant reduction in their sperm count and circulating testosterone levels (Figure , F and G). Testes, epididymal, and seminal vesicle weights were also reduced, while circulating LH levels were higher in Ctsk-Cre
mice (Figure , H–K, and Supplemental Figure 8A). Moreover, expression of the genes encoding the major enzymes required for testosterone biosynthesis (StAR
was decreased in Ctsk-Cre
testes, while expression of HSD-17
was not (Figure L). These phenotypic and molecular abnormalities mimic what is seen in Osteocalcin–/–
male mice (2
), suggesting that osteoclastic bone resorption is necessary for normal male fertility by increasing osteocalcin bioactivity. To add support to this notion, we treated Ctsk-Cre
transplanted mice with recombinant osteocalcin (3 ng/g of BW, 30 days). At the end of this treatment period, the defects in testes, epididymides, and seminal vesicle weights, sperm count, and circulating testosterone levels observed in Ctsk-Cre
transplanted mice had all been corrected (Supplemental Figure 4, B–D). The detrimental effects of irradiation on spermatogenesis and testis function could certainly explain the lower values of the data presented in Figure .
Second, to add credence to the notion that bone resorption is a determinant of the ability of osteocalcin to regulate male fertility, we next studied a gain of function model. Osteoprotegerin (OPG) is a decoy receptor inhibiting the function of the osteoclast differentiation factor RANKL, and both Opg+/–
mice are osteoporotic because of a vast increase in the number of osteoclasts (19
). We noted that there was in Opg–/–
mice a massive increase in the undercarboxylated (GLU13), i.e., active form of osteocalcin (Figure A). A smaller increase of undercarboxylated osteocalcin was also observed in Opg+/–
mice. Consequently, testes, epididymides, and seminal vesicle weights, sperm count, and circulating testosterone levels were all increased in Opg–/–
mice and to a lesser extent in Opg+/–
(Figure , B–F, and Supplemental Figure 8B), and expression of StAR
was increased in Opg+/–
testes while expression of HSD-17
was not (Figure G). Finally, circulating LH levels were lower in Opg+/–
compared with WT serum.
An increase in osteoclast number favors both osteocalcin activity and male fertility.
Third, to more firmly establish that it is the increase in undercarboxylated osteocalcin that explains the male reproduction phenotype of Opg-deficient mice, we took advantage of the fact that the Opg mutation is dominant and therefore that Opg+/– mice have an increase in bone resorption and in male fertility parameters (Figure ). Hence, we asked what would be the consequences of deleting 1 allele of Osteocalcin from Opg+/– mice (Opg+/–;Osteocalcin+/–). The circulating levels of the undercarboxylated form of osteocalcin that were high in Opg+/– were normalized in Opg+/–;Osteocalcin+/– male mice (Supplemental Figure 5). Taken together, results gathered from the analysis of loss- and gain-of-function models of bone resorption indicate that, in the mouse, bone resorption is a physiological determinant of osteocalcin’s regulation of male fertility through its ability to activate osteocalcin.
Insulin signaling in osteoblasts favors testosterone biosynthesis in the mouse.
The cardinal role of bone resorption in the regulation of male fertility provided an opportunity to look for additional upstream regulators of osteocalcin reproductive function. Since it is a positive regulator of bone resorption and it promotes reproductive function, we tested here the hypothesis that insulin signaling in osteoblasts might influence testosterone biosynthesis in an osteocalcin-dependent manner.
For that purpose, we analyzed 12-week-old male mice lacking the gene encoding for insulin receptor selectively in osteoblasts (InsRosb–/–
). These animals that have lower circulating levels of active osteocalcin demonstrated a decrease in testes size and weight, in epididymides and seminal vesicle weights, in sperm count and circulating testosterone levels (Figure , A–F). Moreover, and as is the case in Osteocalcin–/–
male mice, expression of StAR
was decreased in InsRosb–/–
testes, while HSD-17
expression was not (Figure G). Thus the phenotypic, biochemical, and molecular abnormalities of InsRosb–/–
male mice are indistinguishable from those of Osteocalcin–/–
male mice (2
). These results implied that, as is the case for metabolism, insulin signaling in osteoblasts favors testosterone biosynthesis by increasing osteocalcin bioactivity.
Insulin signaling in osteoblasts favors testosterone production.
To demonstrate that it is indeed the case, we next generated compound mutant mice lacking 1 allele of InsR
in osteoblasts and 1 allele of either Osteocalcin
. As shown in Figure , A–E, and Supplemental Figure 8, C and D, whether we looked at testis, epididymides, and seminal vesicle weights or sperm count and circulating levels of testosterone, these compound mutant mice demonstrated abnormalities that were similar to those seen in InsRosb–/–
, or, even more relevantly, in Gprc6a–/–
). Taken together, these results point toward the existence of a pancreas-bone-testis axis in the control of male fertility that acts in parallel to the hypothalamus-pituitary-testis axis.
Insulin signaling in osteoblasts promotes male fertility in an osteocalcin-dependent manner.
Identification of missense mutation in GPRC6A.
In the second part of this study, we addressed another critical aspect of osteocalcin biology, namely, its relevance to humans. To date, the only direct, i.e., genetic evidence that Osteocalcin is a hormone has been obtained in mice. However, the biological importance of osteocalcin would be greatly enhanced if it could be shown to have conserved its function in humans as well.
To address this question, we looked at a patient populations with a well-defined phenotype. The reproductive phenotype of male Osteocalcin–/–
mice is characterized by subfertility in the confined environment of a mouse facility, mediocre spermogram, low circulating testosterone levels, and high circulating LH levels (2
). This presentation is similar to that of peripheral testicular insufficiency in humans (20
). Thus, we performed a genomic analysis of patients harboring similar reproductive abnormalities to Osteocalcin–/–
mice with the goal of identifying loss-of-function mutation in Osteocalcin
that would explain their clinical presentation. We are aware that GPRC6A may have several ligands in vitro; however, the only one for which there is genetic evidence that it binds to this receptor is osteocalcin.
Out of 1,700 new patients seen in an outpatient clinic for fertility disorders, we obtained DNA samples of 59 male patients aged from 32.2 to 49.4 years who had been diagnosed for infertility with oligospermia (sperm count less than 5 million/ml) and high LH circulating levels, but without a corresponding increase in circulating testosterone levels. We used these 3 parameters as a definition of primary testicular failure (20
). All tested patients had a normal karyotype (14
). We sequenced all exons of Osteocalcin
, the receptor mediating osteocalcin reproductive function in Leydig cells (2
), in these patients. Two patients in this cohort harbored a T→A transversion in exon 4 (g.117121904A/T), resulting in an amino acid substitution in a transmembrane domain of GPRC6A (F464Y) (Figure , A and B, and Supplemental Figure 6A). Cross-species alignment showed that the F464 position is conserved down to zebra fish (Figure , A and B, and Supplemental Figure 6A). Accordingly, the F464Y substitution is considered to be deleterious by all prediction programs tested: Polyphen2 (probably damaging, score = 1), SIFT (not tolerated), PhD-SNP (disease related, RI = 6), and Panther (P deleterious = 0.96).
Identification of an amino acid substitution of GPRC6A associated with decreased fertility in humans.
Next, we sequenced as controls 278 individuals originating from Senegal and Togo, the countries of origin of the patients with the GPRC6A mutation, all with normal spermogram, and did not find any harboring this mutation. We also sequenced the exon 4 of GPRC6A in 664 additional male controls, who were either African American, of mixed European descent, or Asian and also failed to find this mutation in any of them. Hence, we found this mutation in 2 out of 59 patients (3.39%) and in none of the 942 controls (Fisher exact test P = 0.036 vs African controls and P = 0.003 vs. all controls). We pursued our analysis and also searched public databases for this single nucleotide variant: the F464Y substitution was detected in 52/4352 African-American and 2/8598 (0.02%) European chromosomes sequenced in the NHLBI GO Exome Sequencing Project, a study whose principal aim is to discover novel genes underlying cardiovascular disorders. These data suggest that F464Y is a rare allele of GPRC6A.
The first patient was a 45-year-old man originating from Togo, with no medical records except that indicating an inability to procreate. His parents were deceased; however, we were able to test his sister, who did not harbor the mutation described below (Supplemental Figure 6B). The patient presented a testicular volume decreased by 6 ml for the right side and 5 ml for the left. Semen analysis showed an oligospermia with 2.71 million/ml (normal >40), normal vitality with 6% necrospermia, low bioavailable testosterone levels (1.4 nmol/l; normal range = 2.3–10.7 nmol/l), elevated LH (12 UI/l; normal range = 0.5–10 UI/l) and follicle-stimulating hormone (FSH) levels (between 26.7–32.6 UI/l; normal range = 1.3–11.5 UI/l), and decreased inhibin B (<15 ng/l) (Table ). The patient had normal bone mass and bone microarchitecture as assessed by HR-pQCT (Scanco) and normal circulating levels of osteocalcin (Table ).
Hormonal and metabolic profiles of the patient presenting an F464Y amino acid substitution of GPRC6A
The similarity between the clinical presentation of this patient and the phenotype of Osteocalcin–/–
mice extended further. Indeed, this patient presented a metabolic syndrome characterized by an increase in BMI (31.6 kg/mβ [Nl < 25 kg/mβ]), elevated waist circumference (107 cm), an increase in adiposity at 28.1% as assessed by whole-body densitometry, and dyslipidemia as well as glucose intolerance determined by hyperinsulinemia after fasting, a glucose tolerance test, and an insulin tolerance test (Tables and ). Many of these features are seen in mice lacking Osteocalcin
in all cells (2
Oral glucose tolerance test performed in the patients presenting an F464Y amino acid substitution of GPRC6A
The second patient was a 35 year-old man originating from Senegal (Tables and , and Supplemental Figure 6C) who sought medical advice after 4 attempts of intracytoplasmic sperm injection failed to result in pregnancy. He has a history of high glucose blood levels that is kept under control by intensive daily exercise and a strict caloric restriction (Tables and , and Supplemental Figure 6C). The patient reported a left orchidectomy for cryptorchidism at 8 years of age. Right testicular volume was 18 ml. Semen analysis showed an extreme oligospermia with 0.87 million/ml (normal > 40); vitality was decreased with 21% necrospermia. Relatively low levels of bioavailable testosterone (4.7 nmol/l; normal range = 2.3–10.7 nmol/l) in the face of elevated LH levels (17 UI/l; normal range = 0.5–10 UI/l) confirmed the diagnosis of primary testicular failure (Table ). FSH levels were slightly elevated at 7.23 UI/l (normal range = 1.3–11.5 UI/l) (Table ). To rule out the possibility that his oligospermia and sterility were a direct consequence of his unilateral cryptorchidism, we compared his sperm count to those of the 9 control individuals who also had orchidectomy due to unilateral cryptorchidism. As presented in Supplemental Table 1, the sperm count in these controls was an average of 17.8 million/ml, i.e., 20-fold higher than in this patient. Physical examination noted the following: weight, 79 kg; height, 1.78 m (BMI = 25 kg/mβ); and blood pressure, 120/70 mmHg (see Tables and ).
The F464Y amino acid substitution in GPRC6A acts as a dominant negative mutation.
When examined in cell culture, the F464Y substitution-mutation resulted in a loss of function of GPRC6A. First, when HEK293T cells were transfected with this mutated form of GPRC6A, osteocalcin stimulation failed to increase cAMP production as it did in cells transfected with WT GPRC6A (Figure ). Second, immunofluorescence of HEK293T cells transfected with either a WT or a F464Y mutated MYC-tagged version of GPRC6A showed that this mutation prevented the localization of the receptor to the cell membrane, which instead accumulated in the endoplasmic reticulum (Figure C). Osteocalcin also failed to increase cAMP production in cells cotransfected with equal amounts of expression vector for the WT and F464Y mutated forms of GPRC6A (Figure D). Western blot analysis confirmed that similar amounts of the WT and F464Y mutated forms of GPRC6A were present in HEK293T cells transfected with each cDNA (Supplemental Figure 7A).
Further experiments suggested that this F464Y substitution in GPRC6A acts as a dominant negative mutation. Indeed in cotransfection experiments using a fixed amount of the WT form of GPRC6A and increasing amounts of its mutated form, cAMP production was significantly decreased whether cotransfections were performed at a ratio of 1:0.25 (WT/mutated), 1:0.5, 1:0.75, or 1:1 (Figure E). Western blot and quantitative PCR verified the different levels of expression of the mutated and WT forms of GPRC6A at the different ratios (Figure F). Immunofluorescent staining on transfected HEK293T cells showed that the WT form of GPRC6A never reached the cell membrane when it was cotransfected with the mutated form of GPRC6A (Figure G). Instead, it remained localized with the F464Y mutant around the nucleus of cells cotransfected at the ratio of 1:1 (Figure G) and 1:0.25 (Figure H).
To add further support to the notion that the F464Y mutation in GPRC6A acts as a dominant negative mutation, we transfected WT and the F464Y GPRC6A in TM3 cells. We observed that StAR
expression was increased by osteocalcin in TM3 Leydig cells overexpressing WT GPRC6A
, but not in TM3 cells overexpressing the F464Y mutant (Figure I). Finally, WT mice were injected intratesticularly with a lentivirus expressing either WT GPRC6A
or the F464Y mutant. As a control, the contralateral testis of each mouse was injected with the vehicle. Three weeks later, the expression levels of 3
, and Cyp11a
were analyzed by qPCR. As shown in Figure J, WT testes injected with vehicle or with WT GPRC6A lentivirus demonstrate a level of expression similar to that in these 3 genes, while expression of these genes was significantly reduced in the testes injected with the lentivirus expressing the F464A mutated form of this receptor. This decrease in 3
, and Cyp11a
was of the same severity if not more severe than that observed in Gprc6a
-deficient testis (2
), suggesting that the F464Y mutation acts as a dominant negative in vivo.
Taken together, results of these 3 different cell culture and in vivo assays are consistent with the notion that the F464Y substitution in GPRC6A may be a cause of the primary testicular failure observed in these 2 patients.