Targeted disruption of the mouse testis-enriched gene Znf230 does not affect spermatogenesis or fertility

Abstract

The mouse testis-enriched Znf230 gene, which encodes a type of RING finger protein, is present primarily in the nuclei of spermatogonia, the acrosome and the tail of spermatozoa. To investigate the role of Znf230 in spermatogenesis, we generated Znf230-deficient mice by disrupting Znf230 exon-5 and exon-6 using homologous recombination. The homozygous Znf230-knockout (KO) mice did not exhibit Znf230 mRNA expression and Znf230 protein production. Znf230 KO mice exhibited no obvious impairment in body growth or fertility. Male Znf230 KO mice had integral reproductive systems and mature sperm that were regular in number and shape. The developmental stages of male germ cells of Znf230 KO mice were also normal. We further examined variations in the transcriptomes of testicular tissue between Znf230 KO and wild-type mice through microarray analysis. The results showed that the mRNA level of one unclassified transcript 4921513I08Rik was increased and that the mRNA levels of three other transcripts, i.e., 4930448A20Rik, 4931431B13Rik and potassium channel tetramerisation domain containing 14(Kctd14), were reduced more than two-fold in Znf230 KO mice compared with wild-type mice. Using our current examination techniques, these findings suggested that Znf230 deficiency in mice may not affect growth, fertility or spermatogenesis.

Keywords: Kctd14; Znf230; knockout mice; spermatogenesis.

Figure 1

The targeted knockout strategy for the Znf230 gene: wild-type allele, targeting construct and targeted allele with the location of primers. Primer pairs P1F/P1R and P2F/P2R, respectively, which flanked the targeted region, were used to select positive ESC clones, and were used to monitor the inheritance of the mutant allele. Primers WTf and WTr were used to amplify the wild-type Znf230 allele. Primers Nf and Nr were used to amplify the knockout allele.

Figure 2

The generation and identification of Znf230 KO mice. (A) L-PCR analysis using primer pairs P1F/P1R and P2F/P2R to amplify the targeted Znf230 alleles from genomic DNA extracted from ESCs. ESCs from No. A7, E4 and H3 were the positive clones undergoing targeted homologous recombination. (B) L-PCR analysis of the targeted Znf230 alleles amplified from genomic DNA derived from the offspring of chimeric mice backcrossed to C57BL/6J. No. 1–10: the offspring members and N: negative control. (C) L-PCR analysis to monitor the inheritance of the targeted Znf230 allele in the progeny of heterozygous Znf230 KO mice. (D) PCR analysis to monitor the inheritance of the targeted Znf230 allele in the progeny of heterozygous Znf230 KO mice using primer pairs WTf/WTr and Nf/Nr. (E) RT-PCR analysis of the Znf230 gene in testes from Znf230 KO and C57BL/6J mice. M1, M2: DNA ladders of 1 kb and 100 bp, respectively. Gapdh was used as an internal control. (F) Western blot analysis of the Znf230 protein in the testes from Znf230 KO and C57BL/6J mice. β-actin was used as an internal control.

Figure 3

Characteristics comparison of reproductive organs and sperm shape between Znf230 KO and C57BL/6J mice. Morphology of reproductive organs from Znf230 KO (A) and C57BL/6J (B) mice. The seminal vesicles (black arrows), bladder (black arrowheads), epididymis (white arrows) and testes (red arrows) were highlighted. Scale bar = 1 cm. Histological analysis of H&E stained testes from Znf230 KO (C,E,G) and C57BL/6J (D,F,H) mice. SG: Spermatogonia, SC: Spermatocyte, RS: Round spermatids, and LS: elongated spermatids. Scale bar = 100 μm. Characteristics of H&E stained sperm from Znf230 KO (I) and C57BL/6J (J) mice.. Ac: acrosome, T: sperm tail. Scale bar = 50 μm.

Figure 4

Comparative qRT-PCR analysis of mRNA levels of the Kctd14 gene between Znf230 KO and C57BL/6J (WT) mice. Bars represent the means ± S.D., Statistical p-values < 0.001.