MEIG1 is essential for spermiogenesis in mice

Abstract

Spermatogenesis can be divided into three stages: spermatogonial mitosis, meiosis of spermatocytes, and spermiogenesis. During spermiogenesis, spermatids undergo dramatic morphological changes including formation of a flagellum and chromosomal packaging and condensation of the nucleus into the sperm head. The genes regulating the latter processes are largely unknown. We previously discovered that a bi-functional gene, Spag16, is essential for spermatogenesis. SPAG16S, the 35 kDa, testis-specific isoform derived from the Spag16 gene, was found to bind to meiosis expressed gene 1 product (MEIG1), a protein originally thought to play a role in meiosis. We inactivated the Meig1 gene and, unexpectedly, found that Meig1 mutant male mice had no obvious defect in meiosis, but were sterile as a result of impaired spermatogenesis at the stage of elongation and condensation. Transmission electron microscopy revealed that the manchette, a microtubular organelle essential for sperm head and flagellar formation was disrupted in spermatids of MEIG1-deficient mice. We also found that MEIG1 associates with the Parkin co-regulated gene (PACRG) protein, and that testicular PACRG protein is reduced in MEIG1-deficient mice. PACRG is thought to play a key role in assembly of the axonemes/flagella and the reproductive phenotype of Pacrg-deficient mice mirrors that of the Meig1 mutant mice. Our findings reveal a critical role for the MEIG1/PARCG partnership in manchette structure and function and the control of spermiogenesis.

Fig. 1.

Expression patterns of the three Meig1 isoforms. (A) Structure of the Meig1 gene. Solid boxes indicate translated exons (1 and 2), open, notched, and crossed boxes indicate nontranslated exons (1a, 1b, and 1c). The arrows indicate location of RT-PCR primers. The red solid boxes indicate 3′UTR of the Meig1 transcripts. I, II, III under 1b represent three consecutive sequences in the exon. (B) Tissue distribution of the three Meig1 transcripts. H: heart; B: brain; SP: spleen; Lu: lung; Li: liver; Sm: skeletal muscle; K: kidney; T: testis; Oc: oocyte; Ov: oviduct. (C) mRNA expression of the three Meig1 transcripts during the first wave of spermatogenesis (RT-PCR analysis).

Fig. 2.

Phenotype of Meig1/CMV-Cre mutant mice. (A) Representative PCR results using a primer set (P3 and P4 in Fig. S3A, primer sequences in Table S1) showing heterozygous (+/−), wild-type (+/+), and homozygous (−/−) genotypes. (B) Northern blot analysis of testicular Meig1 mRNA expression in wild-type, heterozygous and homozygous mice with Meig1 full-length cDNA as the probe. (C) All of the three Meig1 transcripts are disrupted in all of the tissues in Meig1/CMV-Cre mice as revealed by PCR using isoform specific primers sets. B: brain; Li: liver; Lu: lung; Sp: spleen; K: kidney; H: heart; T: testis. (D) Western blot analysis of testicular MEIG1 protein expression in wild-type, heterozygous and homozygous mice. (E) Representative testicular H&E staining images from wild-type and Meig1 homozygous mutant mice. (F) Analysis of haploid (D), diploid (2D), and tetraploid cells (4D) cell populations in wild-type and knockout mice by flow cytometry. (G) Representative images of epididymides from a wild-type mouse (+/+) and a Meig1 homozygous mutant mouse (−/−). (H) Representative sperm collected in 2 mL PBS from cauda epididymides from a wild-type (+/+) and a Meig1 homozygous (−/−) mice. The arrow points to a sperm with a round head, the arrow head points to a sperm with a detached head. (I) Sperm count of wild-type (+/+), heterozygous (+/−), and homozygous (−/−) mice. n = 8 for each group. *: P < 0.001.

Fig. 3.

Testicular ultrastructure in adult wild-type and Meig1 mutant mice. Representative transmission electronic microscopy images from an adult wild-type mouse (A–D) and a Meig1 homozygous mutant mouse (E–L). A and B show normal spermatogenesis and axoneme structure in wild-type testes, C and D shows normal condensing spermatids heads and manchette structure (arrow heads) in wild-type testes. E shows failure of spermatogenesis as evaluated by absence of sperm in the lumen of seminiferous tubule. F represents highly condensed Sertoli cell cytoplasm (arrow). G and H represent some condensing spermatids that lack manchette structure as seen in the wild-type testes (C and D). Inserts in F and G represent two deformed sperm heads. I–L and inserts show disorganized flagella. Note that the flagella components, such as microtubules and outer dense fibers seem to be made normally, but are not assembled correctly into flagella. Some flagella contain multiple axonemal structure (J). Arrows point to disorganized flagella.

Fig. 4.

MEIG1 associates with PACRG. (A) Expression of PACRG in COS-1 cells. COS-1 cells were transfected with the PACRG/pEGFP-C1 or empty pEGFP-C1 vector. Forty-eight hours after transfection, total cell lysates were prepared and Western blots were performed with an anti-GFP antibody (i) and an anti-PACRG antibody (ii). (B) Co-immuniprecipitation of MEIG1 and PACRG. COS-1 cells were co-transfected with PACRG/pEGFP-C1 and MEIG1/pTarget plasmids. Forty-eight hours after transfection, total cell lysates were pulled down with a preimmune serum or an anti-MEIG1 antibody. Western blots were conducted with an anti-GFP antibody (i) and an anti-MEIG1 antibody (ii). (C) Representative Western blot analysis of testicular PACRG protein expression in Meig1 mutant mice. v/v: quaking^viable mutant mice; v/v TG: Pacrg-rescued qk^v mice. (D) Real-time PCR analysis of Pacrg mRNA expression in Meig1 mutant mice. (E) Representative Western blot analysis of testicular MEIG1 protein expression in Pacrg mutant mice.