Expression of Runx2 transcription factor in non-skeletal tissues, sperm and brain

Abstract

Runx2 is a master transcription factor for chondrocyte and osteoblast differentiation and bone formation. However expression of Runx2 (by RT-PCR), has been reported in non-skeletal tissues such as breast, T cells and testis. To better define Runx2 activity in non-skeletal tissues, we examined transgenic (Tg) mice expressing LacZ gene under control of 3.0 kb (3 kb Tg) or 1.0 kb (1 kb Tg) of the Runx2 distal (P1) promoter, Runx2 LacZ knock-in (Runx2(+/LacZ)) and Runx2/P1 LacZ knock-in (Runx2/P1(+/LacZ)). In the Runx2 3 kb Tg mouse, beta-galactosidase (beta-gal) expression appeared in various non-skeletal tissues including testis, skin, adrenal gland and brain. beta-gal expression from both 3 kb and 1 kb Tg, reflecting activity of the Runx2 promoter, was readily detectable in seminiferous tubules of the testis and the epididymis. At the single cell level, beta-gal was detected in spermatids and mature sperms not in sertoli or Leydig cells. We also detected a positive signal from the Runx2(+/LacZ) and Runx2/P1(+/LacZ) mice. Indeed, Runx2 expression was observed in isolated mature sperms, which was confirmed by RT-PCR and Western blot analysis. Runx2, however, was not related to sex determination and sperm motility. Runx2 mediated beta-gal activity is also found robustly in the hippocampus and frontal lobe of the brain in Runx2(+/LacZ). Collectively, these results indicate that Runx2 is expressed in several non-skeletal tissues particularly sperms of testis and hippocampus of brain. It suggests that Runx2 may play an important role in male reproductive organ testis and brain.

Fig. 1

The β-gal activity in testis of 3 Kg and 1 Kb Runx2 distal promoter transgenic and Runx2^+/LacZ mice. (A) β-gal activity is positive in 3 Kb, 1 Kb and Runx2^+/LacZ mice. Whole tissues of 4-month old mice were stained for overnight with X-gal. (B) Promoter activity of Runx2 of seminiferous tubules in testis. Cryosection tissues (5 sm) of testis were stained with X-gal for overnight. The 3 Kb and 1 Kb Tg mice strongly expressed β-gal activity. Also, the β-gal activity weakly appeared in Runx2^+/LacZ mice. The LacZ expression was positive only in a certain stage of cells during spermatogenesis in all three kinds of mice. Expression of β-gal was observed from round spermatids in 3 Kb, 1 Kb Tg and Runx2^+/LacZ mice. Promoter activity was not observed in Sertoli cells and Leydig cells.

Fig. 1

The β-gal activity in testis of 3 Kg and 1 Kb Runx2 distal promoter transgenic and Runx2^+/LacZ mice. (A) β-gal activity is positive in 3 Kb, 1 Kb and Runx2^+/LacZ mice. Whole tissues of 4-month old mice were stained for overnight with X-gal. (B) Promoter activity of Runx2 of seminiferous tubules in testis. Cryosection tissues (5 sm) of testis were stained with X-gal for overnight. The 3 Kb and 1 Kb Tg mice strongly expressed β-gal activity. Also, the β-gal activity weakly appeared in Runx2^+/LacZ mice. The LacZ expression was positive only in a certain stage of cells during spermatogenesis in all three kinds of mice. Expression of β-gal was observed from round spermatids in 3 Kb, 1 Kb Tg and Runx2^+/LacZ mice. Promoter activity was not observed in Sertoli cells and Leydig cells.

Fig. 2. Expression of β-galactosidase in mature sperms

Promoter activity was strongly detected in epididymis spermatids and sperms in 3 Kb and 1 Kb transgenic mice and Runx2^+/LacZ mice. Cryosection tissues of epididymis of 4-month old mice were stained with X-gal for overnight. S; mature sperm.

Fig. 3

Expression of Runx2 in isolated sperms. (A) Total RNA and proteins were isolated from testis and sperm of 8 weeks old wild type mice for RT-PCR using primers that detect total Runx2 levels. Arrow indicates Runx2 signals as 480 bp. Runx2 expressing ATDC5 chondrocyte was used as positive control. (B) Western blot analysis in wild type mice. Runx2 protein was detected as two major bands in both testis and sperm. (C) Expression of Runx2 isoforms in Runx2/P1^+/LacZ. Real-time PCR was performed as described in materials and methods for Runx1, 2 and 3 expression. P1, Runx2 type II mRNA; P2, Runx2 type I mRNA. * P<0.05.

Fig. 3

Expression of Runx2 in isolated sperms. (A) Total RNA and proteins were isolated from testis and sperm of 8 weeks old wild type mice for RT-PCR using primers that detect total Runx2 levels. Arrow indicates Runx2 signals as 480 bp. Runx2 expressing ATDC5 chondrocyte was used as positive control. (B) Western blot analysis in wild type mice. Runx2 protein was detected as two major bands in both testis and sperm. (C) Expression of Runx2 isoforms in Runx2/P1^+/LacZ. Real-time PCR was performed as described in materials and methods for Runx1, 2 and 3 expression. P1, Runx2 type II mRNA; P2, Runx2 type I mRNA. * P<0.05.

Fig. 4

Sex determination by Sry typing and sperm motility assay. (A) Genotyping for sex determination. Genomic DNA was isolated from whole tissue of paraffin block or tail. PCR analysis was conducted as described in materials and methods. M, 1 Kb plus ladder (Invitrogen). (B) The motility assay of sperms from Runx2^+/LacZ mice of 4-month old mice was not changed. The values are the means ± S.D (N=3 in each group). WT; wild type mouse. HE; heterozygous mouse.

Fig. 4

Sex determination by Sry typing and sperm motility assay. (A) Genotyping for sex determination. Genomic DNA was isolated from whole tissue of paraffin block or tail. PCR analysis was conducted as described in materials and methods. M, 1 Kb plus ladder (Invitrogen). (B) The motility assay of sperms from Runx2^+/LacZ mice of 4-month old mice was not changed. The values are the means ± S.D (N=3 in each group). WT; wild type mouse. HE; heterozygous mouse.

Fig. 5

Runx2 promoter activity in brain. (A) β-galactosidase activity is positive in 3 Kb Tg and Runx2^+/LacZ mice. (B) LacZ positive cells were observed in cerebellum in 3 Kb Tg mice. The β-gal activity was appeared granular layer (GL) but not detected in purkinje cells (arrow). (C) The longitudinal section of brain in Runx2^+/LacZ mice. LacZ positive cells were observed in frontal lobe area (F) and hippocampal area CA1 and CA2 but not in CA3 in Runx2^+/LacZ mice. Whole tissues and cryosection tissues of 4-month old mice were stained for overnight with X-gal.

Fig. 5

Runx2 promoter activity in brain. (A) β-galactosidase activity is positive in 3 Kb Tg and Runx2^+/LacZ mice. (B) LacZ positive cells were observed in cerebellum in 3 Kb Tg mice. The β-gal activity was appeared granular layer (GL) but not detected in purkinje cells (arrow). (C) The longitudinal section of brain in Runx2^+/LacZ mice. LacZ positive cells were observed in frontal lobe area (F) and hippocampal area CA1 and CA2 but not in CA3 in Runx2^+/LacZ mice. Whole tissues and cryosection tissues of 4-month old mice were stained for overnight with X-gal.

Fig. 5

Runx2 promoter activity in brain. (A) β-galactosidase activity is positive in 3 Kb Tg and Runx2^+/LacZ mice. (B) LacZ positive cells were observed in cerebellum in 3 Kb Tg mice. The β-gal activity was appeared granular layer (GL) but not detected in purkinje cells (arrow). (C) The longitudinal section of brain in Runx2^+/LacZ mice. LacZ positive cells were observed in frontal lobe area (F) and hippocampal area CA1 and CA2 but not in CA3 in Runx2^+/LacZ mice. Whole tissues and cryosection tissues of 4-month old mice were stained for overnight with X-gal.