Testis-specific Fank1 gene in knockdown mice produces oligospermia via apoptosis

Abstract

Fank1 is exclusively expressed in the testis from the meiosis phase to the haploid phase of spermatogenesis. In this study, we examined the function of Fank1 by establishing a Fank1-knockdown transgenic mouse model. The apoptotic statuses of the testes of the transgenic mice were tested using the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) method. The FANK1 consensus DNA-binding sequence was identified using cyclic amplification of sequence target (CAST) analysis. Differentially expressed genes were examined using microarray analysis. A reduction in sperm number and an increase in apoptotic spermatocytes were observed in Fank1-knockdown mice, and the apoptotic cells were found to be primarily spermatogonia and spermatocytes. The CAST results demonstrated that the consensus DNA-binding sequence was AAAAAG, in which the percentage occurrence of each base at each position ranged from 55 to 86%. This sequence was present in the promoter regions of 10 differentially expressed genes that were examined using microarray analysis. In total, 17 genes were differentially expressed with changes in their expression levels greater than twofold. The abnormal expression of Fank1 target genes that were regulated directly or indirectly by Fank1 reduced the number of sperm in the knockdown mice. Thus, FANK1 may play a pivotal role in spermatogenesis as a transcription factor.

Figure 1

Identification of the GZF1 binding consensus sequence. (a) GST and GST-FANK1 proteins expressed in bacteria were stained with Coomassie Brilliant Blue. M indicates the protein marker (left panel). The GST-FANK1 fusion proteins were detected by Western blotting with an anti-GST antibody (right panel). (b) Alignment of individual DNA sequences recovered in the cyclic amplification of sequence target (CAST) analysis. The deduced consensus FANK1-binding sequence.

Figure 2

Expression of shRNA using the pSUPER-shFank1 vector induces Fank1 mRNA degradation. (a) shFank 631 and 274 are two oligonucleotides located in the ankyrin and FnIII domains of FANK1. Two oligonucleotides containing sense and antisense 20-nt sequences from the Fank1 coding region (black sequence), an 8-nt spacer sequence that provided a loop structure, and a transcription termination signal of five thymidines (purple sequence) were annealed and inserted downstream of the H1 promoter. (b) Transfection of 293 T cells with 1 mg of the pDsRed-Fank-expressing plasmid together with 2 mg of a plasmid expressing siFank. Fluorescence microscopy showed that shFank1 631 and 247 inhibited RFP expression with efficiencies of 90 and 95%, respectively, 36 h after the 293 T cells were transfected. Scale bars = 100 mm. (c) The results of reverse transcription-polymerase chain reaction (RT-PCR) and Western blots suggest that the Fank1 gene is inhibited at the mRNA and protein levels by Fank1 siRNA (P < 0.05).

Figure 3

Identification and phenotype analysis of Fank1 knockdown transgenic mice. (a) In total, 14 founders of Fank1-knockdown mice, eight males and six females, were studied. F₀21, F₀43 and F₀44 were chimeras, F₀20 and F₀24 were not pregnant, and F₀40 and F₀46 died (marked in purple); F₀2, F₀11, F₀12, F₀37, F₀45, F₀1 and F₀39 were studied by phenotype analysis (marked in orange). (b) Expression of Fank1 trangenic mouse siRNAs. Fank1 siRNA expressed in the testes of seven transgenic mice, but not in the wildtype mice. let7a is the blank control. (c) Expression of Fank1 in the testes of transgenic mice and wildtype mice by RT-PCR analyses. Down-regulation of Fank1 in transgenic mice compared with the wildtype mice. Actin is the control. (d) Quantification of the Fank1 transcription level in the testes of seven knock down mice and wildtype male mice using real-time RT-PCR. Expression levels were calculated based on the threshold cycle (Ct) values and were normalized against glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA, and then the expression was determined relative to the level of Fank1. The experiments were repeated at least three times. Data were determined by one-way ANOVA with S-N-K's post-test. (*P < 0.05, **P < 0.01, compared with wildtype mice). (e) Expression of FANK1 protein in the testes of transgenic mice and wildtype mice by Western blot analyses (mean ± s.e.m.). Differences in the FANK1 protein levels between the Fank1-knockdown mice and wildtype mice were observed. Data were determined by one-way ANOVA with S-N-K's post-test (*P < 0.05, **P < 0.01, compared with wildtype mice). ANOVA, analysis of variance; s.e.m., standard error of the mean.

Figure 4

TUNEL staining and confirmation of microarray analysis. (a) and (b) TUNEL staining shows that left and right testis paraffin sections indicate wildtype and Fank1-knockdown mice, respectively. The arrow indicates apoptotic cells. Scale bars = 100 mm. TUNEL staining revealed that the average number of apoptotic cells per tubule cross section in each group was 45.3 ± 7.5 and 121.3 ± 6.3 in the knockdown mice and the wildtype mice, respectively (mean ± s.e.m., P < 0.05, n = 3, t-test), and most of the apoptotic cells were spermatogonia and spermatocytes. (c) Expression of Dusp1 and KlK1b212in Fank1-knockdown mice and wildtype mice by RT-PCR; the 1^st and 2^nd line indicate the Fank1-knockdown mice and wildtype mice, respectively. Dusp1 and KlKlb212 were overexpressed in Fank-knockdown mice compared to the wildtype mice (mean ± s.e.m., P < 0.05, t-test). TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling.