Osteocalcin improved Spermatogenesis in Azoospermic Mouse Model

Tayebeh Rastegar, Pedram Shafaat, Seyed Mohammad Hosein Noori Mougahi, Soraya Parvari, Somayeh Solhjo, Heidar Tooli, Mahsa Yaahoobi Nejad, Akanji Omotosho Dhulqarnain


Infertility is one of the major health concerns in the world whose globally incidence rate is on the rise. Several hormones are effective in this process, such as testosterone, estrogen and Osteocalcin (OCN). The Osteocalcin has Gprc6a receptors on leydig cells, which promotes the production of testosterone when mounted on these receptors. Therefore, the aim of this study was to investigate the role of Osteocalcin on the improvement of spermatogenesis and expression of Gprc6a receptors on leydig cells in the azoospermic mouse model.

Twenty-five mice (4 to 6 weeks) were randomly divided into five groups: control, sham I Group that was initially injected by busulfan solvent (DMSO) at 5 weeks old and then by Osteocalcin solvent (PBS) after 5 weeks for one month, azoospermia Group that received busulfan (40 mg/kg/ip) at 5 weeks old. Sham II Group received busulfan 40mg/kg/ipandt hen after 5 weeks received PBS and experimental group, including azoospermic mice, was administered by Osteocalcin (3ng/g/d) for one month. After the last injection, the tests were dissected and then exposed to the tissue passage. To measure morphological changes, H & E staining was performed on a number of sections to measure the diameter of the seminiferous tubule, thickness of the germinal layer, count the spermatogonial cells, spermatocyte, round spermatid, long spermatid, Sertoli, leydigandmyoid cells. Image J software was used to conduct quantitative studies. Immunohistochemical method was employed to examine the expression of the specific receptor of Osteocalcin, Gprc6α, in the Leydig cell among the groups.

The H & E staining showed a significant difference in the count the spermatogonial cells, spermatocytes, round spermatids, long spermatids, the thickness of the germ layer, the seminiferous tubule diameter between the studied groups (P<0.05).There was also no significant difference in the count of Sertoli, leydig, myoid cells and seminiferous tubule diameter between the groups (P<0.05). In the immune histochemistry, no significant difference was found in the count of GpRC6α-positive leydig cells between the groups (P< 0.05).According to the current results, the OCN plays an important role in spermatogenesis, which has a positive effect on the count of spermatocytes and spermatids, and can be further explored as an appropriate therapeutic strategy proposed for the infertility. 


Osteocalcin, Spermatogenesis, Azoospermia, Leydig Cell, Gprc6α

Full Text:



Zegers-Hochschild F, Adamson GD, de Mouzon J, Ishihara O, Mansour R, Nygren K, et al. The international committee for monitoring assisted reproductive technology (ICMART) and the world health organization (WHO) revised glossary on ART terminology, 2009. Human reproduction. 2009;24(11):2683-7.

Hodžić A, Ristanović M, Zorn B, Tulić C, Maver A, Novaković I, et al. Genetic variation in circadian rhythm genes CLOCK and ARNTL as risk factor for male infertility. PloS one. 2013;8(3):e59220.

Blomberg Jensen M. Vitamin D and male reproduction. Nat Rev Endocrinol. 2014;10(3):175-86.

Smith LB, Saunders PT. The skeleton: the new controller of male fertility? Cell. 2011;144(5):642-3.

Schwetz V, Gumpold R, Graupp M, Hacker N, Schweighofer N, Trummer O, et al. Osteocalcin is not a strong determinant of serum testosterone and sperm count in men from infertile couples. Andrology. 2013;1(4):590-4.

Oury F. A crosstalk between bone and gonads. Annals of the New York Academy of Sciences. 2012;1260(1):1-7.

Holdcraft RW, Braun RE. Androgen receptor function is required in Sertoli cells for the terminal differentiation of haploid spermatids. Development. 2004;131(2):459-67.

Yokonishi T, Ogawa T. Cryopreservation of testis tissues and in vitro spermatogenesis. Reproductive medicine and biology. 2016;15(1):21-8.

Khosla S, Riggs BL. Pathophysiology of age-related bone loss and osteoporosis. Endocrinology and Metabolism Clinics. 2005;34(4):1015-30.

Brinster CJ, Ryu B-Y, Avarbock MR, Karagenc L, Brinster RL, Orwig KE. Restoration of Fertility by Germ Cell Transplantation Requires Effective Recipient Preparation 1. Biology of reproduction. 2003;69(2):412-20.

Pi M, Wu Y, Quarles LD. GPRC6A mediates responses to osteocalcin in β‐cells in vitro and pancreas in vivo. Journal of Bone and Mineral Research. 2011;26(7):1680-3.

Zhou B, Li H, Liu J, Xu L, Zang W, Wu S, et al. Intermittent injections of osteocalcin reverse autophagic dysfunction and endoplasmic reticulum stress resulting from diet-induced obesity in the vascular tissue via the NFκB-p65-dependent mechanism. Cell Cycle. 2013;12(12):1901-13.

Ahar NH, Khaki A, Akbari G, Novin MG. The Effect of Busulfan on Body Weight, Testis Weight and MDA Enzymes in Male Rats. Intern J Women’s Heal and Reprod Sc. 2014;2:316-19.

Björndahl L, Söderlund I, Kvist U. Evaluation of the one‐step eosin‐nigrosin staining technique for human sperm vitality assessment. Human reproduction. 2003;18(4):813-6.

Bancroft JD, Layton C. The Hematoxylin and eosin. Theory & Practice of histological techniques 7th ed, Churchill Livingstone of El Sevier, Philadelphia. 2013:173-214.

Oury F, Ferron M, Huizhen W, Confavreux C, Xu L, Lacombe J, et al. Osteocalcin regulates murine and human fertility through a pancreas-bone-testis axis. The Journal of clinical investigation. 2013;123(6):2421.

Oury F, Sumara G, Sumara O, Ferron M, Chang H, Smith CE, et al. Endocrine regulation of male fertility by the skeleton. Cell. 2011;144(5):796-809.

Pi M, Chen L, Huang M-Z, Zhu W, Ringhofer B, Luo J, et al. GPRC6A null mice exhibit osteopenia, feminization and metabolic syndrome. PLoS One. 2008;3(12):e3858.

Pi M, Faber P, Ekema G, Jackson PD, Ting A, Wang N, et al. Identification of a novel extracellular cation-sensing G-protein-coupled receptor. Journal of Biological Chemistry. 2005;280(48):40201-9.

Wellendorph P, Hansen KB, Balsgaard A, Greenwood JR, Egebjerg J, Bräuner-Osborne H. Deorphanization of GPRC6A: a promiscuous L-α-amino acid receptor with preference for basic amino acids. Molecular pharmacology. 2005;67(3):589-97.

De Toni L, Di Nisio A, Speltra E, Rocca MS, Ghezzi M, Zuccarello D, et al. Polymorphism rs2274911 of GPRC6A as a novel risk factor for testis failure. The Journal of Clinical Endocrinology & Metabolism. 2016;101(3):953-61.

Karsenty G, Oury F. Regulation of male fertility by the bone-derived hormone osteocalcin. Molecular and cellular endocrinology. 2014;382(1):521-6.

Kirmani S, Atkinson EJ, Melton LJ, Riggs BL, Amin S, Khosla S. Relationship of testosterone and osteocalcin levels during growth. Journal of Bone and Mineral Research. 2011;26(9):2212-6.

Kanazawa I, Tanaka K, Ogawa N, Yamauchi M, Yamaguchi T, Sugimoto T. Undercarboxylated osteocalcin is positively associated with free testosterone in male patients with type 2 diabetes mellitus. Osteoporosis International. 2013;24(3):1115-9.

Legrand E, Hedde C, Gallois Y, Degasne I, De Casson FB, Mathieu E, et al. Osteoporosis in men: a potential role for the sex hormone binding globulin. Bone. 2001;29(1):90-5.

Limonard EJ, Schoor N, Jongh R, Lips P, Fliers E, Bisschop PH. Osteocalcin and the pituitary‐gonadal axis in older men: a population‐based study. Clinical endocrinology. 2015;82(5):753-9.

El‐Kamshoushi A, Hassan E, Hassaan P. Evaluation of serum level of Osteocalcin hormone in male infertility. Andrologia. 2017;49(9).

Ferron M, Lacombe J. Regulation of energy metabolism by the skeleton: osteocalcin and beyond. Archives of biochemistry and biophysics. 2014;561:137-46.

Overvad S, Bay K, Bojesen A, Gravholt C. Low INSL3 in Klinefelter syndrome is related to osteocalcin, testosterone treatment and body composition, as well as measures of the hypothalamic–pituitary–gonadal axis. Andrology. 2014;2(3):421-7.

DOI: https://doi.org/10.23954/osj.v3i1.1530


  • There are currently no refbacks.

Creative Commons License
This work is licensed under a Creative Commons Attribution 4.0 International License.

Open Science Journal (OSJ) is multidisciplinary Open Access journal. We accept scientifically rigorous research, regardless of novelty. OSJ broad scope provides a platform to publish original research in all areas of sciences, including interdisciplinary and replication studies as well as negative results.