Osteocalcin Expresses In Leydigcell And Function Research

Bone is one of the most important force-bearing organs and support the body, movement and mineral storage. Osteocalcin is the most abundant protein in the bone expect type I collagen. Osteocalcin is generally considered as closed relation with bone formation and mineral deposition. However, osteocalcin knockout mice exhibited increased bone mass and strength, which means that osteocalcin may have exactly opposite function. Recent studies showed that bone might have endocrine organ function. Osteocalcin secreted by osteoblast regulated energy metabolism and gonadal function through promoting the synthesis of insulin and testosterone. Studies showed that osteocalcin m RNA could be detected in other tissues outside bone, including in liver, kidney, testis, ovary, pancreas, and other human tissues or cells. Although recent research achieved a major breakthrough about osteocalcin, the function of carboxylated osteocalcin is still not clear. The cell type and function of osteocalcin expression in organ outside bone is still need to be further confirmed.An animal model in which osteocalcin promoter activity could be reflected by luciferase reporter was constructed to screen the organ/tissue with osteocalcin promoter activity. Animal molecular imaging techniques was used to detect luciferase distribution in vivo, observed osteocalcin promoter expression location. The results from molecular imaging in vitro and in vivo showed that osteocalcin promoter was active in testis. The activity of osteocalcin promoter increased with the mouse growth.The expression of osteocalcin gene in mice, rat and other species was analyzed by q PCR, ELISA etc. Using IHC, the cell type which expressing osteocalcin was screened and the results from molecular imaging was confirmed in gene level. Osteocalcin m RNA can be detected in mice, rat, bovine testis. Osteocalcin protein can be detected in mice testis by Elisa. Using IHC, the luciferase was located in Leydig of transgenic mice, so did osteocalcin in wild type mice. Using different primer pair, it was excluded that the detected osteocalcin m RNA is a side production from PMF1 passthrough transcription or ORG. Different concentration of VD3 was added to the medium. Luciferase activity was detected using Luciferase Assay System. Osteocalcin promoter activity and gene expression rise when VD3 was added to Leydig medium, suggest that bone related cytokine like VD3 could regulated osteocalcin expression in Leydig.To explore the function of osteocalcin expressing in Leydig, undercarboxylation osteocalcin was purified from prokaryotic E.Coli and added to culture medium of Leydig cell with different concentrations. The testosterone synthesis ability was detected by q PCR and Elisa to analyze the effects of undercarboxylated osteocalcin. Leydig-specific overexpression osteocalcin gene plasmid was constructed and stably transfected into TM3. Knockdown osteocalcin cell line was constructed using sh RNA plasmid. The expression of testosterone synthesis key enzyme, glucose metabolism enzyme, testosterone synthesis and cell proliferation were analyzed using q PCR, Elisa in the stable cell line and sh RNA-transfected TM3. Addition of exogenous undercarboxylated osteocalcin to Leydig promoted expression of key enzyme in testosterone synthesis. Knockdown of endogenous osteocalcin didn’t significantly affect the testosterone synthesis key enzyme STAR, Cyp11 a expression. Overexpression of osteocalcin in Leydig cells resulted in expression decline of key enzyme in testosterone synthesis, and increased expression of SOX-9 and Runx2. A transgenic mice with Leydig-specific overexpressing osteocalcin was developed. Testis-specific overexpression of osteocalcin leaded to significant decrease of testis weight and testosterone synthesis in RLF-OG-Luc transit mice. SOX-9 gene expression level was raised in testis in transgenic mice testis. Leydig-specific osteocalcin overexpression also induced a decreased diameter of seminiferous tubule, fall of spermatogenic cell and microlithiasis in testis. These results suggest the function of self-expressing osteocalcin was different from the exogenous addition undercarboxylated osteocalcin.To explore the form and function of osteocalcin expressing in Leydig, Warfarin which could specifically block vitamin circulation and carboxylation process was added to the cell lines. Expression alternation of key enzyme in testosterone synthesis was detected by q PCR after adding warfarin to Leydig culture medium. CRISPR-cas9 system was used to develop an vitamin K dependent carboxylase(GGCX) knockout TM3 cell line. The gene related to osteocalcin, glucose metabolism, testosterone synthesis were analysis by q PCR. GGCX expression in testis and Leydig was higher than in bone and osteoblast. In osteocalcin overexpression TM3 cells, GGCX knockout resulted in increased expression of STAR and Cyp11 a, decline of SOX-9 expression. Warfarin treatment blocked the vitamin K dependent carboxylation process and restored the expression of STAR, Cyp11 a to normal level(wild type) in overexpressed-osteocalcin TM3 cell line. Hydroxyapatite could absorb the osteocalcin overexpression TM3 and induced it clustering growth. Above results indicated that the osteocalcin expressed in Leydig was carboxylated.Taken together, this study documented osteocalcin m RNA and protein existed in Leydig cell with its carboxylated type. Overexpression of carboxylated osteocalcin in Leydig cell decreased key enzyme expression of testosterone synthesis and increased SOX-9, Runx2 gene expression, which may increase the risk of testis stone and decrease testosterone synthesis. These results provide new clue for carboxylated osteocalcin research.