Expression of uncharacterized male germ cell-specific genes and discovery of novel sperm-tail proteins in mice

Abstract

The identification and characterization of germ cell-specific genes are essential if we hope to comprehensively understand the mechanisms of spermatogenesis and fertilization. Here, we searched the mouse UniGene databases and identified 13 novel genes as being putatively testis-specific or -predominant. Our in silico and in vitro analyses revealed that the expressions of these genes are testis- and germ cell-specific, and that they are regulated in a stage-specific manner during spermatogenesis. We generated antibodies against the proteins encoded by seven of the genes to facilitate their characterization in male germ cells. Immunoblotting and immunofluorescence analyses revealed that one of these proteins was expressed only in testicular germ cells, three were expressed in both testicular germ cells and testicular sperm, and the remaining three were expressed in sperm of the testicular stages and in mature sperm from the epididymis. Further analysis of the latter three proteins showed that they were all associated with cytoskeletal structures in the sperm flagellum. Among them, MORN5, which is predicted to contain three MORN motifs, is conserved between mouse and human sperm. In conclusion, we herein identify 13 authentic genes with male germ cell-specific expression, and provide comprehensive information about these genes and their encoded products. Our finding will facilitate future investigations into the functional roles of these novel genes in spermatogenesis and sperm functions.

Fig 1. Testis- and germ cell-specific expression of the 13 novel genes.

The tissue distributions of the 13 selected genes were assessed by RT-PCR in eight different tissues of adult male mice. Germ cell-specific expression was also examined in germ cell-lacking testes from W/W^v mutant mice. Glyceraldehyde-3-phosphate dehydrogenase (Gapdh) was amplified as a loading control. WT, wild-type testis; W/W^v, germ cell-less testis. All genes exhibited testis- and germ cell-specific expression.

Fig 2. Developmental expression of the selected genes during spermatogenesis.

Juvenile spermatogenesis consists of the mitotic, meiotic and postmeiotic phases. The meiotic phase includes the preleptotene (PL), leptotene (L), zygotene (Z), pachytene (P), and diplotene (D) stages. Diplotene spermatocytes undergo two meiotic divisions (MI and MII). The stage-specific expressions of the selected genes were determined from mouse testes obtained on days 8, 10, 12, 14, 16, 20, and 30 after birth. Gapdh was included as a loading control. All of the selected genes, with the exception of Mm.159422, were expressed from spermatocytes. Spcy, spermatocytes; Sptd, round spermatids.

Fig 3. In silico analysis of the genomic, transcript, and protein characteristics of the selected genes.

The chromosomal localization and intron-exon organization of each gene were determined by genome database searches. Under ‘exon organization,’ the boxes indicate exons. The bars represent the regions amplified in our PCR analysis. The protein-coding regions (black-shaded) were determined by selecting the longest open reading frames deduced from the predicted cDNA sequences. The position of the poly A signal is marked by an arrowhead, and the presence of poly A is indicated by ‘A.’ The Gene Ontology (GO) terms for each gene product were obtained from the Gene Ontology Consortium; they all fall under the broad categories of molecular function (M), cellular component (C), and biological process (B).

Fig 4. Expression patterns of seven of the novel proteins in various tissues and the postnatal testes of mice.

A. The tissue distributions of some of the novel proteins were examined by immunoblotting, with GAPDH detected as a loading control. All of the gene products showed specific expression in testis. B. The stage-specific expressions of the novel proteins were determined from mouse testes obtained on days 7, 14, 21, 28, 35, 42, 49, and 56 after birth. The anti-α-tubulin antibody was used as a control. All of the proteins were expressed in late meiotic or postmeiotic germ cells.

Fig 5. Developmental expression pattern of the novel proteins.

A. Protein samples from testicular cells (TC), testicular sperm (TS), and mature sperm (S) were blotted with antibodies against the novel proteins. α-Tubulin was detected as a control. The expressions of the selected proteins were found to be developmentally regulated during sperm maturation. B. Sperm from the cauda epididymis and vas deferens were separated into head (H) and tail (T) fractions, and subjected to Western blot analysis. ADAM2 and α-tubulin were used to confirm the head and tail fractions, respectively. All of the selected proteins were found to be expressed in the sperm tail.

Fig 6. Localization of the novel proteins in adult testis.

A. Immunofluorescence staining of paraffin sections of adult testis was conducted using specific antibodies for the four proteins. Normal rabbit serum (NRS) was used as a negative control. The red color indicates proteins and nuclei were stained with DAPI (blue). Images in the boxes are magnified in the insets in merged images. Scale bar, 100 μm. B. Localization of Mm.271255/1700013F07Rik in mature sperm. Sperm from the cauda epididymis and vas deferens were immunostained with anti-Mm.271255/1700013F07Rik. DAPI was used to stain nuclei. Mm.271255/1700013F07Rik is localized to the neck and midpiece of sperm. Scale bar, 100 μm.

Fig 7. Characterization of three sperm tail proteins.

A. Sperm from the epididymis and vas deferens were treated with 1% NP-40 or 1% Triton X-100 and then centrifuged. Soluble and insoluble fractions were subjected to immunoblot analysis. ADAM2 and α-tubulin were used to verify the soluble and insoluble fractions, respectively. The tested proteins failed to solubilize with these detergents. S, supernatant after centrifugation; P, pellet after centrifugation. B. Sperm were treated with 2, 3, 4, or 6 M urea and then centrifuged. Soluble and insoluble fractions were subjected to Western blot analysis, with α-tubulin detected as a loading control. MORN5 and Mm.271255 were found to be insoluble in urea.

Fig 8. Expression of MORN5 in human sperm.

A. Extracts of sperm from mouse and humans were subjected to SDS-PAGE and blotted with anti-MORN5. Tubulin was detected as a loading control. MORN5 was expressed in human sperm. B. Diagram of human and mouse MORN5, showing amino acid identities and domain information. The predicted MORN5 proteins are 170 and 161 amino acids in mouse and human, respectively, and contain three MORN motifs (gray boxes). The antigenic region used to raise the mouse MORN5 antibody is indicated by a red line. This region showed 85% identity between the mouse and human protein sequences (whole protein: 79% identity).