Mammalian Fused is essential for sperm head shaping and periaxonemal structure formation during spermatogenesis

Abstract

During mammalian spermatogenesis, the diploid spermatogonia mature into haploid spermatozoa through a highly controlled process of mitosis, meiosis and post-meiotic morphological remodeling (spermiogenesis). Despite important progress made in this area, the molecular mechanisms underpinning this transformation are poorly understood. Our analysis of the expression and function of the putative serine-threonine kinase Fused (Fu) provides critical insight into key steps in spermatogenesis. In this report, we demonstrate that conditional inactivation of Fu in male germ cells results in infertility due to diminished sperm count, abnormal head shaping, decapitation and motility defects of the sperm. Interestingly, mutant flagellar axonemes are intact but exhibit altered periaxonemal structures that affect motility. These data suggest that Fu plays a central role in shaping the sperm head and controlling the organization of the periaxonemal structures in the flagellum. We show that Fu localizes to multiple tubulin-containing or microtubule-organizing structures, including the manchette and the acrosome-acroplaxome complex that are involved in spermatid head shaping. In addition, Fu interacts with the outer dense fiber protein Odf1, a major component of the periaxonemal structures in the sperm flagellum, and Kif27, which is detected in the manchette. We propose that disrupted Fu function in these structures underlies the head and flagellar defects in Fu-deficient sperm. Since a majority of human male infertility syndromes stem from reduced sperm motility and structural defects, uncovering Fu׳s role in spermiogenesis provides new insight into the causes of sterility and the biology of reproduction.

Keywords: Fertility; Fused; Kif27; Manchette; Periaxonemal; Sperm.

Figure 1. Conditional deletion of Fused in the germ cells leads to reduced sperm count despite normal morphology of testes and epididymides

(A–C, F–H) Isotopic in situ hybridization using ^33P-UTP-labeled riboprobes (pink) derived from Fu, Kif27, Spag16 on paraffin sections of wild-type (A–C) and Vasa-Fu (F–H) mouse adult testes. Fu, Kif27, Spag16 were all expressed in the germ cells residing in the seminiferous tubules. Vasa-Fu testes in which Vasa-Cre converted a conditional allele of Fu into a null allele showed no Fu expression (F). Kif27 and Spag16 expressions in the testes were unaltered in the absence of Fu. (D, E, I, J) Hematoxylin and eosin-stained sections of testes (D, I) and cauda epididymis (E, J) from wild-type and Vasa-Fu adult mice. The seminiferous tubules appeared morphologically normal in the absence of Fu and were populated by germ cells and Sertoli nurse cells. Fu-deficient cauda epididymis where mature spermatozoa are stored was also morphologically normal. However, a decrease in spermatozoa density was detected in cauda epididymides of Vasa-Fu mice. Scale bar = 100 µm for A–C and F–H; 50 µm for D, E, I, J. (K, L) Whole-mount β-gal (LacZ) staining of testes and epididymides from R26R and Vasa-Cre; R26R mice at postnatal (p) day 1. Specific β-gal staining was observed in the developing seminiferous tubules of the Vasa-Cre; R26R testes in which Cre expression activated a β-gal reporter from the R26R locus.

Figure 2. Vasa-Fu spermatozoa have disrupted head morphology associated with perturbed manchette formation and function

(A, D) Phase contrast images of mature spermatozoa derived from the cauda epididymis of wild-type and Vasa-Fu adult mice. Vasa-Fu flagella were of normal length and contained all the main structures (head, midpiece and flagellum). (B, E) Hematoxylin and eosin-stained spermatozoa derived from the cauda epididymis of wild-type and Vasa-Fu adult mice. The nucleus stained blue. The Vasa-Fu sperm head was elongated and lacked the characteristic hook shape in wild-type mouse spermatozoa heads. (C, F) Transmission electron micrographs of wild-type and Vasa-Fu mature spermatozoa from the testes. Wild-type sperm heads (C) showed a tight association between the nucleus (Nu) (black, electron-dense) and the acrosome cap (Ac) (dark grey). By contrast, the sperm head from Vasa-Fu (F) had a large gap between the nucleus and the acrosome cap. (G–V) Transmission electron micrographs of wild-type and Vasa-Fu spermatids. (G, O) Cap phase spermatid (steps 4–7). The round spermatids are characterized by the establishment of the acrosome/acroplaxome complex at one end of the nucleus. (H–J, P–R) Acrosome phase spermatids (steps 8–14). The manchette was discerned by microtubules on either side of the nucleus. Alteration in the nuclear shape, gaps in the acrosome/acroplaxome complex and excessive elongation of the manchette were observed in elongating Vasa-Fu spermatids. (K, L, S, T) Maturation phase spermatids (steps 15–19). The nucleus was fully condensed and elongated. The annulus (two large dots) began to migrate to form the midpiece. In Vasa-Fu spermatids, the nucleus and the perinuclear ring were distorted (S) (arrow). Thinning of the extended nucleus and fragmentation of the membranes could also be seen (T) (arrow). (M, U) High-resolution view of the connecting piece in an elongating spermatid. Note the large vesicles (arrow) at the base of the nucleus in the Vasa-Fu spermatid (U). (N, V) High-resolution view of the manchette microtubules in wild-type and Vasa-Fu spermatids. The microtubules appeared more abundant and were also disordered and asymmetrical in the mutants (compare black lines in V to N). (W–Z) Immunostaining of wild-type germ cells derived from the testes at various stages of spermiogenesis (steps 8–16 shown). The manchette was marked by anti-acetylated (Ac) tubulin antibodies (red) while the nucleus was stained with DAPI (blue). The flagellum was also labeled by Ac-tubulin (Z). (E’–H’) Immunostaining of Vasa-Fu germ cells derived from the testes at similar stages. The manchette structure appeared around the nucleus (E’) but became disorganized subsequently throughout spermiogenesis (F’–H’). Ectopic microtubules and defective nuclear head shaping were noticeable (F’–H’). Scale bar = 5 µm for W–Z, E’–H’. (A’–D’, I’–L’) Schematic representation of manchette (red) development in relation to the nucleus (black) during spermiogenesis in wild-type and Vasa-Fu spermatids. In Vasa-Fu mutant spermatids, the manchette is abnormally elongated and also present ectopically and the nucleus is misshapen.

Figure 3. Testicular Vasa-Fu sperm flagella display periaxonemal abnormalities

(A–L) Transmission electron micrographs of sperm from wild-type and Vasa-Fu adult mouse testes. Cross sections through the midpiece, principal piece and end piece were shown. In the principal piece, large gaps and vesicles (arrows in H, I) were observed in the transverse ribs of the fibrous sheath with unidentified material within. In more distal sections, an additional longitudinal column (LC) (arrows in J, K) that surrounds the axoneme was detected. The LCs are characterized by electron-dense material anchored to a microtubule doublet of the axoneme. Scale bar = 0.1 µm for A, G. Scale bar = 0.1 µm for B–F, H–L. (M–R) Distribution of longitudinal columns in wild-type and Vasa-Fu flagella derived from the testes. The numbers (1–9) refer to the position of microtubule doublets; the LCs surround microtubule doublets at positions 8 and 3 in wild-type sperm (also see S). An extra LC was detected at position 6 in (O), 9 in (P) and 4 in (Q). Approximately 5% of axonemes examined had two additional longitudinal columns (R). Scale bar = 0.1 µm for M–R. (S) Schematic diagram of mouse sperm. The head is connected to the flagellum by a connecting piece, from which nine outer dense fibers (ODFs) originate from the head-tail coupling apparatus (HTCA) and extend into the flagellum. The flagellum is characterized by a “9+2” motile axoneme in which nine microtubule doublets surround a central pair. The doublets are numbered clockwise starting with the doublet on the plane that bisects the central pair. Each ODF surrounds a microtubule doublet in the midpiece. The principal piece is covered by the fibrous sheath (FS) that contains two longitudinal columns, each replacing an ODF (at microtubule doublet positions 8 and 3). The longitudinal columns are associated with a specific microtubule doublet through a longitudinal anchor (LC-A). The columns are connected by keratinous transverse ribs and are proposed to maintain the structural integrity of the flagellum and affect the plane of flagellar motion and the waveform of the beat. The periaxonemal structures provide stability and strength to the flagellum.

Figure 4. FuGFP and Kif27GFP localize to various microtubule-organizing centers of the developing sperm

(A–D, I–P) Confocal immunofluorescence of testicular cells derived from Vasa-FuGFP transgenic mice where a Fu-GFP fusion protein was expressed under the germ cell-specific Vasa promoter. Cells were stained with antibodies against GFP (green) and acetylated tubulin (red) while the nucleus was stained with DAPI (blue). FuGFP was seen in the intercellular bridges (A) on the plasma membrane of round spermatids. The signal in the intercellular bridges persisted through the elongation steps of spermiogenesis. In spermatids with condensing chromatin (B, C), FuGFP was found in the manchette, the perinuclear ring and the acrosome-acroplaxome. In elongated spermatids (D), the manchette disappeared and the FuGFP signal was reduced. (E–H) Schematic diagram illustrating the localization of FuGFP (green) during spermiogenesis. (I–L) Additional stages of Vasa-FuGFP during spermiogenesis. Spermatocytes had broad cytoplasmic FuGFP staining (I). FuGFP staining was present in the manchette of a round spermatid (J) and in the developing acrosome-acroplaxome (J–L). (M–P) A step 12/13 elongating Vasa-FuGFP spermatid displayed immunofluorescence in the acrosome-acroplaxome, perinuclear ring (arrow) and manchette. (Q–T) Confocal immunofluorescence of testicular cells derived from wild-type adult mice. Cells were stained with antibodies against STK36 (green) and acetylated (Ac) tubulin (red); the nucleus was stained with DAPI (blue). STK36 signal was primarily detected in the perinuclear ring of round (Q), elongating (R, S) and elongated (T) spermatids. Weaker STK36 staining was also present in the manchette and the acrosome-acroplaxome (S). (U–X) Schematic diagram illustrating the localization of endogenous Fu (Stk36) during spermiogenesis. Not drawn to scale. (Y–B’) Confocal immunofluorescence of testicular cells derived from Vasa-Fu adult mice. STK36 staining was residual and non-specific. (C’–F’) Confocal immunofluorescence of testicular cells derived from Vasa-Kif27GFP transgenic mice. GFP signal was detected in the cytoplasm of round spermatids (C’). In elongating spermatids (D’, E’), specific signal was detected in the manchette and along the perinuclear ring that borders it. Kif27GFP signal translocated to the head-tail coupling apparatus (HTCA) at later stages (F’). After spermiation, no GFP signal was detected in the spermatozoa for either FuGFP or Kif27GFP. Scale = 5 µm for A–D, I–T, Y–B’. (G’–J’) Schematic diagram illustrating the localization of Kif27GFP (green) during spermiogenesis. Not drawn to scale. IB, intercellular bridge; M, manchette; AAC, acrosome-acroplaxome complex, PNC, perinuclear ring, HTCA, head-tail coupling apparatus.

Figure 5. Fu interacts with the heat-shock family protein Odf1

(A) Western blot of immunoprecipitated Fu^FLAG and deletion mutants of Fu (FuΔN^FLAG and FuΔC^FLAG) to test physical interactions between Fu and Odf1 using HEK293T cell lysates. Odf1^Myc was pulled down from the cell lysate when Fu^FLAG or FuΔC^FLAG (lacking the C-terminal regulatory domain) was immunoprecipitated. However, Odf1^Myc failed to be co-immunoprecipitated with FuΔN^FLAG that lacks the kinase domain. (B) Western blot of immunoprecipitated Fu^FLAG or Odf1^Myc from HEK293T cell lysates to examine their physical interactions. Fu^Myc can be pulled down when Odf1^FLAG was immunoprecipitated with anti-FLAG agarose. In a control experiment, Odf1^FLAG failed to bring down Sufu^Myc (not shown). In, input; IP, immunoprecipitation; WB, Western blot. The numbers indicate locations of protein size standards.