Acroframosome-dependent KIFC1 facilitates acrosome formation during spermatogenesis in the caridean shrimp Exopalaemon modestus

Abstract

Background: Acrosome formation and nuclear shaping are the main events in spermatogenesis. During spermiogenesis in Exopalaemon modestus, a unique microtubular structure called the acroframosome (AFS) forms in spermatids. The AFS links to a temporary organelle called the lamellar complex (LCx) leading to the formation of an everted umbrella-shaped acrosome and a dish-shaped nucleus in the mature sperm. These morphological changes require complex cell motility in which the C-terminal kinesin motor protein called KIFC1 is involved. In this study, we demonstrate that KIFC1 moves along the AFS and plays an important role in acrosome formation and nuclear shaping during spermatogenesis in E. modestus.

Methodology/principal findings: We cloned a 3125 bp complete cDNA of kifc1 from the testis of E. modestus by PCR. The predicted secondary and tertiary structures of E. modestus KIFC1 contain three domains: a) the C-terminus, b) the stalk region, and the c) N-terminusl. Semi-quantitative RT-PCR detected the expression of kifc1 mRNA in different tissues of E. modestus. In situ hybridization demonstrated the temporal and spatial expression profile of kifc1 during spermiogenesis. Western blot identified the expression of KIFC1 in different tissues of E. modestus, including the testis. Immunofluorescence localized KIFC1, tubulin, GM130, and mitochondria in order to elucidate their role during spermiogenesis in E. modestus.

Conclusion/significance: Our results indicate that KIFC1 transports the Golgi complex, mitochondria, and other cellular components that results in acrosome formation and nuclear shaping in E. modestus. The KIFC1 transport function depends upon the microtubular structure called the acroframosome (AFS). This study describes some of the molecular mechanisms involved in the acrosome formation and nuclear shaping in E. modestus. In addition, this study may provide a model for studying the molecular mechanisms involved in spermatogenesis in other crustacean species and lead to a better understanding of the fertilization process in crustaceans.

Figure 1. Full-length cDNA of the kifc1 in E. modestus.

The deduced amino acid sequence is shown above the nucleotide sequence. This figure shows the full-length cDNA of the kifc1 that consists of a 73 bp 5′untranslated region, a 952 bp 3′untranslated region and a 2100 bp open reading frame that encodes 700 amino acids.

Figure 2. Comparison of the KIFC1 protein in E. modestus with its homologues.

This figure shows the amino acid alignment of KIFC1 with its homologues using Vector NTI10 (Invitrogen, California, USA). The AYGQTGSGKT, SSRSH, and LAGSE sequences (red frame) are the putative ATP-binding motifs. The YNETIRDLL sequence (blue frame) is the microtubule-binding motif. The ELKGNIRVFCRVRP sequence (black frame) is the KIFC conserved consensus. The sequenced KIFC1 shows a 69.9, 47.8, 47.9, 49.2, and 46.9% identity with its counterparts in Eriocheir sinensis, Crassostrea gigas, Xenopus laevis, Mus musculus, and Danio rerio, respectively.

Figure 3. The phylogenetic tree of KIFC1 protein and its homologues.

This figure shows the phylogenetic tree of KIFC1 and it homologues that were constructed by the neighbor-joining method using Mega 5 (version 5.0) software. The KIFC1 homologues from Eriocheir sinensis, Gallus gallus, Xenopus laevis, Homo sapiens, Danio rerio, Salmo salar, Rattus norvegicus, and Cynops orientalis were examined. The putative E. modestus KIFC1 protein is most closely related to Eriocheir sinensis.

Figure 4. The major structural features of E. modestus KIFC1.

(A) This figure shows the three structural domains of KIFC1. The C-terminus (346–700 aa) contains the conserved head (yellow bar) that “walks” along the microtubules. The stalk region (154–346 aa) forms an extended coiled-coil region (pink bar). The N-terminus (1–154 aa) contains the divergent tail (blue bar) that carries different cargoes. (B) This figure shows the putative 3-D structure of KIFC1. The head, stalk, and tail are labelled by different colors. See the website http://zhanglab.ccmb.med.umich.edu/I-TASSER/output/S123537/ for more details.

Figure 5. Semi-quantitative RT-PCR analysis of kifc1 gene in different tissues.

(A) This figure shows the kifc1 expression in various E. modestus tissues (upper panel). β-actin was used as a positive control (lower panel). kifc1 is highly expressed in the E. modestus testis. (B) This figure shows a quantitative analysis of kifc1 expression in various E. modestus tissues. kifc1 is highly expressed in the E. modestus testis and hepatopancreas. T: testis, H: heart, M: muscle, He: hepatopancreas and G: gill.

Figure 6. In situ hybridization of kifc1 mRNA during the E. modestus spermiogenesis.

(A,B,I) Early stage of spermiogenesis. These figures show that kifc1 mRNA signals (arrows; blue signal; red dots) are weakly distributed in the cytoplasm of round or oblong spermatids. (C,D,J) Middle stage of spermiogenesis. These figures show that kifc1 mRNA signals (arrows; blue signal; red dots) are increased compared to the early stage and concentrate on one side of the spermatid where the acrosome will eventually form. (E,F,K) Late stage of spermiogenesis. These figures show that kifc1 mRNA signals (arrows; blue signal; red dots) are strongly distributed in the cytoplasm, centralized over the acrosome cap, and localized in a dot-like pattern on the surface of the dish-shaped nucleus. The AFS-derived spike shows a minimal signal. (G,H,L) Mature sperm. These figures show that kifc1 mRNA signals (arrows; blue signal; red dots) are weakly distributed over the acrosome cap but, in general, are dramatically decreased compared to other stages of spermiogenesis. The AFS-derived spike and the dish-shaped nucleus show minimal signal. N: nucleus, M: mitochondria, G: Golgi complex, LCX: lamellar complex, AFS: acroframosome, MF: microfilaments.

Figure 7. Western blot analysis of E. modestus tissues.

The E. modestus tissue extracts were probed with anti-KIFC1 polyclonal antibody (upper panel) and anti-β-actin polyclonal antibody (lower panel) which served as the control. This figure shows that KIFC1 protein is present at the highest levels in the muscle (M), intermediate levels in the testis (T) and heart (H), and the lowest level in the gill (G). The molecular weight of KIFC1 (78 kDa) and β-actin (42 kDa) is shown at the right.

Figure 8. Immunofluorescent localization of tubulin and KIFC1 during the early stage of E. modestus spermiogenesis.

(A) DAPI nuclear staining. (B) Tubulin staining. Tubulin (green staining; arrows) localizes in the cytoplasm and near the nuclear membrane. (C) KIFC1 staining. KIFC1 (red staining; arrows) also localizes in the cytoplasm and near the nuclear membrane. (D). Phase contrast microscopy. This figure shows the general morphological appearance of the spermatid during the early stage of spermiogenesis. (E) Merged immunofluorescent images. Tubulin (green staining; arrows) and KIFC1 (red staining; arrows) co-localize in the cytoplasm and near the nuclear membrane. Nucleus (blue staining). (F) Diagram. This diagram shows the distribution of tubulin (green dots) and KIFC1 (red dots) in the spermatid during the early stage of spermiogenesis. N: nucleus.

Figure 9. Immunofluorescent localization of tubulin and KIFC1 during the middle stage of E. modestus spermiogenesis.

(A) DAPI nuclear staining. (B) Tubulin staining. Tubulin (green staining; arrows) localizes in sporadic areas within the lamellar complex (LCx). (C) KIFC1 staining. KIFC1 (red staining; arrows) localizes throughout the entire area of the LCx. (D) Phase contrast microscopy. This figure shows the general morphological appearance of the spermatid during the middle stage of spermiogenesis. (E) Merged immunofluorescent images. Tubulin (green staining; arrows) and KIFC1 (red staining; arrows) co-localize in relationship with the LCX. Nucleus (blue staining). (F) Diagram. This diagram shows the distribution of tubulin (green dots) and KIFC1 (red dots) in the spermatid during the middle stage of spermiogenesis. N: nucleus, LCx: lamellar complex, MT: microtubules, C: centriole.

Figure 10. Immunofluorescent localization of tubulin and KIFC1 during the late stage of E. modestus spermiogenesis.

(A) DAPI nuclear staining. (B) Tubulin staining. Tubulin (green staining; arrows) localizes specifically to the acroframosome (AFS). (C) KIFC1 staining. KIFC1 (red staining; arrows) localizes mainly to the proacrosome with some staining associated with the AFS. (D) Phase contrast microscopy. This figure shows the general morphological appearance of the spermatid during the late stage of spermiogenesis. (E) Merged immunofluorescent images. Tubulin (green staining; arrows) and KIFC1 (red staining; arrows) localization is shown. Nucleus (blue staining). (F) Diagram. This diagram shows the distribution of tubulin (green) and KIFC1 (red dots) in the spermatid during the late stage of spermiogenesis. N: nucleus, LCx: lamellar complex, AFS: acroframosome, C: centriole.

Figure 11. Immunofluorescent localization of tubulin and KIFC1 in mature E. modestus sperm.

(A) DAPI nuclear staining. (B) Tubulin staining. Tubulin (green staining; arrows) localizes specifically to the center of the acrosome. (C) KIFC1 staining. KIFC1 (red staining; arrows) localizes throughout the whole acrosome including the AFS-derived spike (D) Phase contrast microscopy. This figure shows the general morphological appearance of the E. modestus mature sperm. (E) Merged immunofluorescent images. Tubulin (green staining; arrows) and KIFC1 (red staining; arrows) localization is shown. Nucleus (blue staining). (F) Diagram. This diagram shows the distribution of tubulin (green) and KIFC1 (red dots) in the E. modestus mature sperm. N: nucleus, MF: microfilaments AFS: acroframosome, C: centriole.

Figure 12. Morphological features of the manchette during rat spermatogenesis.

(A,B) Electron microscopy. (A) This electron micrograph shows the absence of the manchette at the start of acrosome formation in rat spermatogenesis. N: nucleus, PA: proacrosome (B) This electron micrograph shows the presence of the manchette (arrows) during nuclear shaping in rat spermatogenesis. N: nucleus (C,D) Immunofluorescent staining. These immunofluorescent light micrographs show the localization of tubulin (green staining) and KIFC1 (red staining) in rat spermatids. The manchette appears to surround the nucleus in a skirt-shaped manner.

Figure 13. Immunofluorescent localization of GM130 (Golgi complex) during E. modestus spermiogenesis.

(A–C) Early stage of spermiogenesis. (A) DAPI staining. (B) GM130 staining. GM130 (green staining; arrows) localizes in the cytoplasm near the periphery of the nucleus. (C) Merged immunofluorescent images. (D,E,F) Middle stage of spermiogenesis. (D) DAPI staining. (E) GM130 staining. GM130 (green staining; arrows) localizes near the LCx and along one side of the nucleus as the nucleus gradually deforms. (F) Merged immunofluorescent images. (G,H,I) Late stage of spermiogenesis. (G) DAPI staining. (H) GM130 staining. GM130 (green staining; arrows) localizes in the LCx and in the proacrosome near the AFS. (I) Merged immunofluorescent images. (J,K,L) Mature stage. (J) DAPI staining. (K) GM130 staining. GM130 (green staining; arrows) localizes in the acrosome. (L) Merged immunofluorescent images.

Figure 14. Immunofluorescent localization of MitoTracker (mitochondria) during E. modestus spermiogenesis.

(A–C) Early stage of spermiogenesis. (A) DAPI staining. (B) MitoTracker staining. MitoTracker (green staining; arrows) localizes in the cytoplasm near the periphery of the nucleus. (C) Merged immunofluorescent images. (D,E,F) Middle stage of spermiogenesis. (D) DAPI staining. (E) MitoTracker staining. MitoTracker (green staining; arrows) localizes near the LCx and along one side of the nucleus as the nucleus gradually deforms. (F) Merged immunofluorescent images. (G,H,I) Late stage of spermiogenesis. (G) DAPI staining. (H) MitoTracker staining. MitoTracker (green staining; arrows) localizes in the LCx and in the proacrosome near the AFS. (I) Merged immunofluorescent images. (J,K,L) Mature stage. (J) DAPI staining. (K) MitoTracker staining. MitoTracker (green staining; arrows) localizes in the acrosome. (L) Merged immunofluorescent images.

Figure 15. The distribution model of GM130 (Golgi complex) and MitoTracker (mitochondria) staining during E. modestus spermiogenesis.

The GM130 and the MitoTracker staining patterns are concordant so they are both represented by orange dots. (A) Early stage of spermiogenesis. (A) Early stage of spermiogenesis. GM130 and MitoTracker localize in the cytoplasm near the periphery of the nucleus. (B) Middle stage of spermiogenesis. GM130 and MitoTracker localize near the LCx and along one side of the nucleus as the nucleus gradually deforms. (C,D) Late stage of spermiogenesis. GM130 and MitoTracker localize in the LCx and in the proacrosome near the AFS, (E) Mature stage. GM130 and MitoTracker localize in the acrosome. M: mitochondrion, N: nucleus, C: centriole, MT: microtubule, MF: microfilament, LCx: lamellar complex, AFS: acroframosome.

Figure 16. The mechanism of KIFC1 participation in acrosome formation during E. modestus spermiogenesis. T

his model shows that KIFC1 moves along the AFS and transports Golgi complex vesicles, mitochondria, and probably other cellular components to fuse into the LCx. KIFC1 transports LCx components along the AFS in order to facilitate acrosome formation during E. modestus spermiogenesis. KIFC1 may also link to the nuclear envelope and participate in nuclear shaping. AFS: acroframosome, LCx: lamellar complex, MF: microfilament, M: mitochondrion, G: Golgi complex, N: nucleus.