Regulation of murine lactate dehydrogenase C (Ldhc) gene expression

Abstract

Expression of Ldhc begins with the onset of meiosis in male germ cells and continues throughout spermatogenesis. Transcriptional regulatory mechanisms, especially in primary spermatocytes, are poorly described because of the lack of a reliable cell culture system. We constructed mouse transgenics and transfected germ cells in situ to study expression of the testis-specific isozyme of lactate dehydrogenase (LDH). From previous work, we determined that a 100-bp Ldhc core promoter contained potential cis regulatory elements, including a palindrome (-21 to +10), GC box (-70 to -65), and cAMP-responsive element (CRE) sites (-53 to -49, -39 to -35). We provide here the demonstration of a functional role for these sequences by expression of mutated transgenes in vivo. Our results reveal for the first time that mutation of the GC box does not abolish promoter activity, which remains testis-specific. Mutation of GC box or CRE sites resulted in a 73% and 74% reduction in promoter activity, respectively, in a transient transfection of germ cells in vivo by electroporation; the combination of GC box and CRE site mutations eliminates promoter activity. Therefore, we conclude that simultaneous occupancy of the GC box and CRE sites in the core promoter is necessary for full expression of Ldhc in the testis.

FIG. 1

Schematic diagrams illustrating the Ldhc core promoter sequence and DNA constructs. A) The 100-bp core promoter containing a GC box, 2 CRE sites, TATA box, palindromic sequence, and transcription initiation element (INR). B) The transgene construct depicting the Ldhc core promoter sequence from −88 to +12 cloned into pNASSβ for LacZ reporter gene expression, mutation sites for SPmu and PALmu constructs were underlined. C) Constructs used for in vivo electroporation; WT-hRluc was used as a transfection control vector co-injected with other test vectors.

FIG. 2

X-gal stained transgenic testis. A) PALmu transgenic littermate identified as negative by PCR. B) PALmu transgenics. C) SPmu testis. D) Stage-specific staining pattern in seminiferous tubules of a PALmu mouse; epididymal staining is from endogenous β-gal. Original magnification ×10.

FIG. 3

β-galactosidase activity analyses. Average β-gal activity from founder lines PALmu323 and SPmu272 were illustrated with nontransgenic control animals. All readings calibrated as RLU (relative light unit) per μg protein. One-way ANOVA and Dunnett’s Multiple Comparsion Test were performed between testis and somatic tissues. Bar represents means ± SEM, n = 3. *P < 0.05, **P < 0.01 indicates significant difference.

FIG. 4

IHC staining of testis sections from PALmu and SPmu transgenic mice. A) Anti-β-gal staining of PALmu transgenic testis section. B) Anti-β-gal staining in SPmu testis sections. C and D) Nontransgenic sections stained with anti-LDHC and anti-β-gal antibodies respectively. Arrows in A and C indicate zygotene (Z) and pachytene (P) spermatocytes. Reaction product is visible only in pachytene and later stages of spermatogenesis (see insets, magnification ×1200). Original magnification ×400.

FIG. 5

In vivo electroporation. Microinjection of pAcGFP-N1 into seminiferous tubules via the efferent duct, followed by electroporation of surgically exposed testis. Diagram shows GFP expression 24 h after electroporation. Top: Bright field view of seminiferous tubules injected with DNA solution containing 10% trypan blue tracking dye. Middle: Fluorescence microscopy of testis (left) and seminiferous tubules (right). Bottom: Fluorescence in frozen section (left) and paraffin-embedded section (right) showing GFP expression in transfected germ cells. Original magnification ×10 for top and middle panel; original magnification ×100 and ×400 for bottom panel left and right, respectively.

FIG. 6

Analysis of Ldhc promoter activity by in vivo electroporation. Horizontal axis represents the percentage of luciferase activity for each mutated promoter sequence compared to the wildtype promoter. Ldhc promoter sequences extended from −88 to +12 in all constructs. WT, wildtype promoter; PALmu, PAL sequence mutation; SPmu, GC box mutation; CREmu, both CRE sites mutated; TRImu, combined mutation of GC box and both CRE sites. One-way ANOVA and two-tailed, unpaired t-test between groups were performed. Bars with different letters are significantly different (P < 0.01).

FIG. 7

Gel-shift assay demonstrating specific interaction of Ldhc promoter oligos containing GC box (Ldhc-SP) or CRE sequences (Ldhc-CRE) with testis nuclear protein extract (TN). Lanes 1 to 7, oligo Ldhc-SP used as probe; lane 1: Ldhc-SP probe; lane 2: TN + probe; lanes 3–5: TN + probe + 50-fold non-radioactive Ldhc-SP, Ldhc-SPmu, and SP1 consensus, respectively; lanes 6–7: TN + probe + antibody. Lanes 8–14, oligo Ldhc-CRE used as probe; lane 8: Ldhc-CRE; lane 9: TN + probe; lanes 10–12: TN + probe + 50-fold non-radioactive Ldhc-CRE, Ldhc-CREmu1, and CRE consensus, respectively; lanes 13–14: TN + probe + antibody. NS, Nonspecific.