Retention of gene products in syncytial spermatids promotes non-Mendelian inheritance as revealed by the t complex responder

Abstract

The t complex responder (Tcr) encoded by the mouse t haplotype is able to cause phenotypic differences between t and + sperm derived from t/+ males, leading to non-Mendelian inheritance. This capability of Tcr contradicts the concept of phenotypic equivalence proposed for sperm cells, which develop in a syncytium and actively share gene products. By analyzing a Tcr minigene in hemizygous transgenic mice, we show that Tcr gene products are post-meiotically expressed and are retained in the haploid sperm cells. The wild-type allele of Tcr, sperm motility kinase-1 (Smok1), behaves in the same manner, suggesting that Tcr/Smok reveal a common mechanism prone to evolve non-Mendelian inheritance in mammals.

Figure 1.

Tcr transcripts escape the general mechanism of product sharing between syncytial sperm cells and are translated at late stages of spermiogenesis. (A) Schematic drawing of the transgene construct Tg9 comprising the Tcr promoter (Tcr prom), untranslated region, and Myc epitope-tagged Tcr coding region (Tcr CDS), followed by the simian virus polyadenylation signal (SV40pA). (B–D) Cryosections of Tg9/0 (B,C) or wild-type (D) testes hybridized in situ with digoxygenin (DIG)-labeled Smok1-specific (B) or Myc epitope-specific (C,D) probes, showing expression of endogenous Smok1 transcripts in all round spermatids (B), whereas Tg9 transcripts are confined to a subpopulation of round spermatids (C) and not detected in the control (D). Dashed lines indicate the outlines of seminiferous tubules, and white boxes indicate subregions shown at higher magnification on the right. Bar, 100 μm. (E) Schematic view of a seminiferous tubule. (Se) Sertoli cells; (SG) spermatogonia; (SC) spermatocytes; (RS) round spermatids; (SZ) spermatozoa. (F, top panel) Immunofluorescent detection of Myc-Tcr protein using an anti-Myc antibody (α-Myc) on testis cryosections of a Tg9/0 male reveals Tcr protein in the lumen of seminiferous tubules. (Middle panel) β-Tubulin (α-Tubb), a major component of axonemes, indicating flagella, localizes to the same region. (G) In epididymal spermatozoa, Myc-Tcr localizes primarily to the principal piece and nucleus (yellow arrows; the flagellum is indicated by a dashed line). (F,G, bottom panel) Wild-type controls show some nonspecific primary antibody reaction. Nuclei are visualized by DAPI staining (blue). Bar, 50 μm.

Figure 2.

Tcr and Smok1 transcripts occur mainly in nuclear and perinuclear aggregates. Serial optical sections (∼1-μm depth) of testicular cryosections obtained by confocal microscopy visualizing Tg9 (A) or Smok1 (B) transcripts (red) detected by fluorescent in situ hybridization in round spermatids. Selected examples of nuclear (arrowhead) and perinuclear (arrow) RNA aggregates are indicated. Tg9 transcripts were detected on sections of Tg9/0-derived testes (shown in A) with a DIG-labeled Myc epitope-specific probe, and Smok1 transcripts were detected on sections of wild-type testes with a Smok1-specific probe. Dashed lines denote the nuclear outlines of round spermatids visualized by DAPI staining (blue), and bright blue structures within the nuclei represent nucleoli. Bar, 6.5 μm.

Figure 3.

Tcr protein is retained in the spermatozoa expressing the transgene. (A,B) Ultrathin sections of spermatozoa derived from the cauda epididymis of Tg9/0 or wild-type control (wt) males assayed for Myc-Tcr protein using immunogold-labeled anti-Myc antibodies reveals the localization of Tcr protein at the fibrous sheath (FS) and the ODFs of the principal piece of the flagellum (A) and in sperm nuclei (B). A fraction of Tg9/0-derived sperm nuclei show no or few immunogold particles, like wild-type sperm. White boxes indicate areas shown at higher magnification on the right; for better visualization, immunogold particles are shown as white spots on duplicate panels. (Ax) Axoneme. Bars: A, 500 nm; B, 280 nm. (C) Histograms showing the number of sections of sperm nuclei corresponding to the number of immunogold particles counted, based on the analysis of 202 and 204 sperm nuclei derived from wild-type (wt) or Tg9/0 animals, respectively. In sperm nuclei of hemizygous Tg9/0 males, one part of the distribution is concentrated at low immunogold particle counts, whereas the remainder is widely spread at higher counts (heavy tail distribution).

Figure 4.

Smok1 shows the same pattern of transcriptional expression and post-transciptional control as Tcr. (A) Schematic drawing of the transgene construct TgS, consisting of the Smok1 promoter (Smok1 prom), the Myc epitope-tagged Smok1 coding region (Smok1 CDS), and untranslated regions followed by the polyadenylation signal (Smok1pA). (B) Testis cryosections of TgS/0 (top panel) or wild-type control (bottom panel) males hybridized in situ with a DIG-labeled Myc epitope-specific probe (Myc) show TgS expression in seminiferous tubules of the TgS/0 male in a subpopulation of round spermatids, but not in the control. White boxes indicate the regions shown at higher magnification on the right, and the borders of seminiferous tubules are indicated by dashed lines. Bar, 100 μm. (C) Immunofluorescent detection of Myc-Smok1 protein on cryosections of seminiferous tubules derived from a TgS/0 (left panel) or a wild-type control (right panel) male using anti-Myc antibodies visualizing Myc-Smok1 protein (red) specifically in the lumen filled with flagella of late-stage spermatids. Nuclei are visualized by DAPI staining (blue). Bar, 50 μm. (D) Model depicting a mechanism leading to non-Mendelian inheritance of functionally different alleles (denoted 1 and 2), as well as genetically linked chromosomal regions (shaded area), of a gene involved in sperm function due to post-meiotic expression and retention of the gene products in the haploid spermatids, thus creating functionally different spermatozoa.